During development, compartmentalization of an early embryonic structure produces blocks of cells with distinct properties and developmental potentials. The auditory and vestibular components of vertebrate inner ears are derived from defined compartments within the otocyst during embryogenesis. The vestibular apparatus, including three semicircular canals, saccule, utricle, and their associated sensory organs, detects angular and linear acceleration of the head and relays the information through vestibular neurons to vestibular nuclei in the brainstem. How the early developmental events manifest vestibular structures at the molecular level is largely unknown. Here, we show that LMO4, a LIM-domain-only transcriptional regulator, is required for the formation of semicircular canals and their associated sensory cristae. Targeted disruption of Lmo4 resulted in the dysmorphogenesis of the vestibule and in the absence of three semicircular canals, anterior and posterior cristae. In Lmo4-null otocysts, canal outpouches failed to form and cell proliferation was reduced in the dorsolateral region. Expression analysis of the known otic markers showed that Lmo4 is essential for the normal expression of Bmp4, Fgf10, Msx1, Isl1, Gata3, and Dlx5 in the dorsolateral domain of the otocyst, whereas the initial compartmentalization of the otocyst remains unaffected. Our results demonstrate that Lmo4 controls the development of the dorsolateral otocyst into semicircular canals and cristae through two distinct mechanisms: regulating the expression of otic specific genes and stimulating the proliferation of the dorsolateral part of the otocyst.

Haploinsufficiency of Gata3 causes hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome in mice and humans. Gata3 null mutation leads to early lethality around embryonic day (E)11.5, but catecholamine precursor administration can rescue Gata3 null mutants to E16.5. At E11.5, GATA3 deficiency results in the development of an empty otocyst with an endolymphatic duct. However, using rescued mice we found that some morphogenesis and neurosensory development is possible in the ear without Gata3. Extending previous studies, we find that at E16.5, Gata3 mutant inner ears can undergo partial morphogenesis and develop an endolymphatic duct, a utricular and saccular recess, and a shortened cochlear duct. In addition to the obvious morphogenic aberrations, these studies demonstrate that a subset of neurons develop and connect a fragmented sensory patch of MYO7A-positive hair cells to the vestibular nuclei of the brainstem. In situ hybridization studies reveal altered expression of several transcription factors relevant to ear development and we hypothesize that this may relate to the observed dysmorphia and restricted neurosensory development. While a cochlear duct can form, there is no concurrent cochlear neurosensory development, observations consistent with specific hearing defects encountered by HDR patients and mice with Gata3-associated expression alterations. Gata3 null mutant phenocopies the otic maldevelopment (cochlear duct formation in the absence of neurosensory development) seen in Foxg1cre mediated conditional deletion of microRNA processing enzyme, Dicer1. Finally, while GATA3 is expressed in the developing vestibulo-cochlear efferent (VCE) neurons, and its absence in the null mutants disrupts VCE projections to the ear, loss of GATA3 does not affect VCE progenitor cell migration.

Patients with HDR syndrome suffer from hypoparathyroidism, deafness, and renal dysplasia due to a heterozygous deletion of the transcription factor GATA3. Since GATA3 is prominently expressed in both the inner ear and different parts of the auditory nervous system, it is not clear whether the deafness in HDR patients is caused by peripheral and/or central deficits. Therefore, we have created and examined heterozygous Gata3 knockout mice. Auditory brainstem response (ABR) thresholds of alert heterozygous Gata3 mice, analyzed from 1 to 19 months of age, showed a hearing loss of 30 dB compared to wild-type littermates. Neither physiological nor morphological abnormalities were found in the brainstem, cerebral cortex, the outer or the middle ear. In contrast, cochleae of heterozygous Gata3 mice showed significant progressive morphological degeneration starting with the outer hair cells (OHCs) at the apex and ultimately affecting all hair cells and supporting cells in the entire cochlea. Together, these findings indicate that hearing loss following Gata3 haploinsufficiency is peripheral in origin and that this defect is detectable from early postnatal development and maintains through adulthood.

Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3(+)CD25(+) T cells, an effect that was reversible by IFN-alpha treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3(+)CD25(+) T cells is dependent on TGF-beta but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3(+)CD25(+) T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3(+)CD25(+) T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-alpha treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.

In a previous study, we reported that intrathecal injection of mesenchymal stem cells (MSCs) slowed disease progression in G93A mutant superoxide dismutase1 transgenic mice. In this study, we found that intrathecal MSC administration vastly increased the infiltration of peripheral immune cells into the spinal cord of Amyotrophic lateral sclerosis (ALS) mice (G93A mutant superoxide dismutase1 transgenic). Thus, we investigated the immunomodulatory effect of MSCs on peripheral blood mononuclear cells (PBMCs) in ALS patients, focusing on regulatory T lymphocytes (Treg ; CD4(+) /CD25(high) /FoxP3(+) ) and the mRNA expression of several cytokines (IFN-γ, TNF-α, IL-17, IL-4, IL-10, IL-13, and TGF-β). Peripheral blood samples were obtained from nine healthy controls (HC) and sixteen patients who were diagnosed with definite or probable ALS. Isolated PBMCs from the blood samples of all subjects were co-cultured with MSCs for 24 or 72 h. Based on a fluorescence-activated cell sorting analysis, we found that co-culture with MSCs increased the Treg /total T-lymphocyte ratio in the PBMCs from both groups according to the co-culture duration. Co-culture of PBMCs with MSCs for 24 h led to elevated mRNA levels of IFN-γ and IL-10 in the PBMCs from both groups. However, after co-culturing for 72 h, although the IFN-γ mRNA level had returned to the basal level in co-cultured HC PBMCs, the IFN-γ mRNA level in co-cultured ALS PBMCs remained elevated. Additionally, the levels of IL-4 and TGF-β were markedly elevated, along with Gata3 mRNA, a Th2 transcription factor mRNA, in both HC and ALS PBMCs co-cultured for 72 h. The elevated expression of these cytokines in the co-culture supernatant was confirmed via ELISA. Furthermore, we found that the increased mRNA level of indoleamine 2,3-dioxygenase (IDO) in the co-cultured MSCs was correlated with the increase in Treg induction. These findings of Treg induction and increased anti-inflammatory cytokine expression in co-cultured ALS PBMCs provide indirect evidence that MSCs may play a role in the immunomodulation of inflammatory responses when MSC therapy is targeted to ALS patients. We propose the following mechanism for the effect of mesenchymal stem cells (MSCs) administered intrathecally in amyotrophic lateral sclerosis (ALS): MSCs increase infiltration of peripheral immune cells into CNS and skew the infiltrated immune cells toward regulatory T lymphocytes (Treg ) and Th2 lymphocytes. Treg and Th2 secret anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β. A series of immunomodulatory mechanism provides a new strategy for ALS treatment.

Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor commonly inactivated in glioblastoma multiforme (GBM), but the prognostic significance of PTEN remains controversial. Here, we demon- strate significant prognostic value of combined PTEN mutation and expression for the survival of patients with GBM on the basis of analysis of large-scale cancer genomic data. PTEN nonsense mutations associated with sig- nificantly shorter disease-free survival and overexpression of PTEN protein linked to shorter disease-free and overall survival of patients with GBM. PTEN nonsense mutations correlated with decreased p53 and Gata3 protein levels and increased genomic instability in human GBM tissues. Expression of nonsense PTEN mutant decreased p53 and Gata3 levels, producing increased DNA damage both in vitro and in vivo. Mice carrying xenograft tumors with nonsense PTEN mutant displayed significantly shorter survival. Our data demonstrated the prognostic value of combined PTEN mutation and protein expression for patients with GBM and highlighted distinct biologic effects of nonsense and missense mutations of PTEN.

Group 2 innate lymphoid cells (ILC2s) are important effector cells driving the initiation of type 2 immune responses leading to adaptive T helper 2 (Th2) immunity. Here we show that ILC2s dynamically express the checkpoint inhibitor molecule PD-L1 during type 2 pulmonary responses. Surprisingly, PD-L1:PD-1 interaction between ILC2s and CD4+ T cells did not inhibit the T cell response, but PD-L1-expressing ILC2s stimulated increased expression of GATA3 and production of IL-13 by Th2 cells both in vitro and in vivo. Conditional deletion of PD-L1 on ILC2s impaired early Th2 polarization and cytokine production, leading to delayed worm expulsion during infection with the gastrointestinal helminth Nippostrongylus brasiliensis Our results identify a novel PD-L1-controlled mechanism for type 2 polarization, with ILC2s mediating an innate checkpoint to control adaptive T helper responses, which has important implications for the treatment of type 2 inflammation.

The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71-72%) but low identities to mammalian T-bet genes (41-42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73-88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial (Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-gamma and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression, may allow novel future control measures against disease in fish to be developed.

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Jawless fishes occupy a critical phylogenetic position in understanding the origin of the adaptive immune system. Here, we performed large-scale expressed sequence tag analysis of leukocytes isolated from the inshore hagfish Eptatretus burgeri. Although we found many immunity-related genes such as those involved in lymphocyte or hematopoietic cell signaling and development as well as cytokine and cytokine receptor genes, MHC molecules or antigen receptors were not identified. We characterized two hagfish cDNAs that closely resembled mammalian proteins with essential roles in adaptive immunity, one encoding a GATA3-like molecule and another encoding a Bruton's tyrosine kinase (Btk)-like molecule. The GATA3-like gene of hagfish was equidistant from GATA3 and GATA2 in jawed vertebrates. Similarly, the hagfish Btk-like molecule was not Btk itself, but qualified as a pre-duplicated form of Btk and Bmx in jawed vertebrates. In total, our work provides circumstantial evidence that adaptive immunity is unique to jawed vertebrates.

GATA3 is a zinc-binding transcription factor that regulates the differentiation of many human tissue types, including the mammary gland. In surgical pathology, immunohistochemistry for GATA3 is largely used to support urothelial or breast origin in a carcinoma of unknown origin. GATA3 is sensitive but not entirely specific in this setting. Although GATA3 labeling is highest in estrogen receptor-positive carcinomas, it also labels estrogen receptor-negative carcinomas and thus has particular diagnostic utility in the setting of triple-negative breast carcinomas, which are typically negative for other mammary-specific markers.

In this paper, we show that the transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons.

CD4 T cells can be primarily polarized to differentiate into Th1 or Th2 cells. CD44 is a marker of T cell activation and a property of long-lived memory cells and implicated in cell migration, activation, and differentiation. To date, whether CD44 has a role in regulating Th1-Th2 differentiation has not been determined. In this study, we compared Th1 and Th2 responses in wild-type and CD44-deficient mice in response to sheep RBC and chicken OVA, as well as examined Th1-Th2 differentiation in vivo and in vitro from CD44-sufficient and CD44-deficient naive CD4 T cells. We observed that deficiency of CD44 tended to inhibit Th1 while promoting Th2 differentiation. Furthermore, chimeric studies suggested that CD44 expression by CD4 T cells was essential for such Th2 bias. The regulation by CD44 occurred at the transcription level leading to up-regulated GATA3 and down-regulated T-bet expression in activated CD4 T cells. We also noted that CD44-deficiency could modify the state of dendritic cell subsets to induce a Th2-biased development. Results presented in this study demonstrate for the first time that CD44 participates in the regulation of Th1-Th2 differentiation.

We have used Affymetrix high-density gene arrays to generate a temporal profile of gene expression during differentiation of UB/OC-1, a conditionally immortal cell line derived from the mouse cochlea. Gene expression was assessed daily for 14 days under differentiating conditions. The experiment was replicated in two separate populations of cells. Profiles for selected genes were correlated with those obtained by RT-PCR, TaqMan analysis, immunoblotting, and immunofluorescence. The results suggest that UB/OC-1 is derived from a population of nonsensory epithelial cells in the greater epithelial ridge that have the potential to differentiate into a hair-cell-like phenotype, without the intervention of Math1. Elements of the Notch signaling cascade were identified, including the receptor Notch3, with a transient up-regulation that suggests a role in hair cell differentiation. Several genes showed a profile similar to Notch3, including the transcriptional co-repressor Groucho1. UB/OC-1 also expressed Me1, a putative partner of Math1 that may confer competence to differentiate into hair cells. Cluster analysis revealed expression profiles for neural guidance genes associated with Gata3. The temporal dimension of this analysis provides a powerful tool to study genetic mechanisms that underlie the conversion of nonsensory epithelial cells into hair cells.

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.

Summary Analysis of T-helper cell differentiation to T-helper type 1 (Th1) and Th2 lineages has begun to reveal a complex mechanism whereby transcription factors, enzymes that either deposit or remove covalent modifications from histone tails and DNA methylating enzymes are recruited to cytokine genes. Each resultant cell lineage subsequently displays a programme of transcriptional restrictions that firstly, facilitates expression of a particular subset of signature cytokines and secondly, silences expression of the cytokines normally recognized as being markers of the opposite differentiation limb. Some essential proteins in this differentiative paradigm, such as the transcription factors GATA3 and T-bet, are well studied; however, the types of enzymatic activities that these proteins recruit in order to implement differentiation are more obscure. Recent genome-wide studies of histone modifications have begun to clarify how specific modifications of histones impact upon both transcriptional regulation and chromatin organization. Here we review how this information has enlightened our knowledge of how Th1/Th2 differentiation is orchestrated.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.

Neurological manifestations are present in 5% to 30% of patients with Behçet's disease (BD). Neuro-Behçet's Disease (NBD) is hypothetically caused by T helper (Th) cells, which development is dependent on the expression of lineage-specific transcription factors. Cerebrospinal fluid (CSF) mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 were assessed in 18 NBD patients and 26 controls disease [16 noninflammatory neurological disease (NIND) and 10 headache attributed to Behçet's disease (HaBD)]. Expression of TBX21 (Th1), RORC (Th17) and Foxp3 (Treg) were increased in NBD patients compared to HaBD and NIND patients. EBI3 and Th2-associated GATA3 expressions were found to be decreased (P<0.0001 and P<0.0001) in NBD patients. Analysis of transcription factor ratios, revealed an increase in the RORC/FOXP3 and TBX21/GATA3 ratios in NBD patients (P<0.0001; P<0.0003). Our findings indicate that both Th1 and Th17 mRNA expressions involving a possible impairment of Treg cells. This might play a role in CSF-NBD inflammation, permitting activation of harmful T cell subpopulations. The TBX21/GATA3 and RORC/FOXP3 ratios dysregulations in NBD are consistent with those reported in other inflammatory diseases and indicating the plasticity existing between Th1, Th17 and Treg cells during inflammation.

Naive T helper cells differentiate into functionally distinct effector subsets that drive specialized immune responses. Recent studies indicate that some of the effector subsets have plasticity. Here, we used an EAE model and found that Th17 cells deficient in the transcription factor BCL11B upregulated the Th2-associated proteins GATA3 and IL-4 without decreasing RAR-related orphan receptor γ (RORγt), IL-17, and GM-CSF levels. Surprisingly, abnormal IL-4 production affected Th17 cell trafficking, diverting migration from the draining lymph nodes/CNS route to the mesenteric lymph nodes/gut route, which ameliorated EAE without overt colitis. T helper cell rerouting in EAE was dependent on IL-4, which enhanced retinoic acid (RA) production by dendritic cells, which further induced expression of gut-homing receptors CCR9 and α4β7 on Bcl11b-deficient CD4+ T cells. Furthermore, IL-4 treatment or Th2 immunization of wild-type mice with EAE caused no alteration in Th17 cytokines or RORγt, but diverted T helper cell trafficking to the gut, which improved EAE outcome without overt colitis. Our data demonstrate that Th17 cells are permissive to Th2 gene expression without affecting Th17 gene expression. This Th17 plasticity has an impact on trafficking, which is a critical component of the immune response and may represent a possible avenue for treating multiple sclerosis.

BACKGROUND

Studies of human asthma and of animal models of allergic inflammation/asthma highlight a crucial role for T(H)2 cells in the pathogenesis of allergic asthma. Repressor of GATA (ROG) is a POZ (BTB) domain-containing Kruppel-type zinc finger family (or POK family) repressor. A repressive function to GATA3, a master transcription factor for T(H)2 cell differentiation, is indicated.

OBJECTIVE

The aim of this study was to clarify the regulatory roles of ROG in the pathogenesis of T(H)2-driven allergic diseases, such as allergic asthma.

METHODS

We examined allergic airway inflammation and airway hyperresponsiveness (AHR) in 3 different mouse models, which use either ROG-deficient (ROG(-/-)) mice, ROG transgenic mice, or adoptive transfer of cells.

RESULTS

In ROG(-/-) mice T(H)2 cell differentiation, T(H)2 responses, eosinophilic airway inflammation, and AHR were enhanced. In ROG transgenic mice the levels of eosinophilic airway inflammation and AHR were dramatically reduced. Furthermore, adoptive transfer of T(H)2 cells with increased or decreased levels of ROG expression into the asthmatic mice resulted in reduced or enhanced airway inflammation, respectively.

CONCLUSIONS

These results indicate that ROG regulates allergic airway inflammation and AHR in a negative manner, and thus ROG might represent another potential therapeutic target for the treatment of asthmatic patients.

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of a member of the GATA-binding family of transcription factors, GATA3. This dual zinc finger transcription factor binds DNA with its C-terminal zinc finger (ZnF2) and stabilizes this binding with its N-terminal zinc finger (ZnF1). ZnF1 also interacts with other zinc finger proteins, notably Friend of GATA (FOG). The HDR syndrome has been described in patients with mutations affecting both ZnF1 and ZnF2 domains; the former result in inefficient interaction with FOG, and the latter result in disruption of DNA binding. We report a patient with renal failure, hypoparathyroidism, and bilateral hearing loss. Assessment of family members indicated that the disease arose as a de novo mutation in her mother. Analysis of GATA3 in the family revealed a heterozygous missense mutation resulting in a nonconservative change of a single amino acid (R276P) in the ZnF1 domain. Functional analysis using dissociation electrophoretic mobility shift and yeast two-hybrid assays showed reduced binding affinity to the GATA motifs but normal interaction with FOG in vitro. These results are consistent with the predicted functions of human GATA3-ZnF1 from three-dimensional molecular modeling and with HDR being a result of GATA3 haploinsufficiency.