Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.

Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.

Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

Variants of the Bach2 gene are linked to vitiligo, celiac disease, and type 1 diabetes, but the underlying immunological mechanisms are unknown. In this study, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence and governing the differentiation, activation, and survival of Foxp3(+) regulatory T (Treg) cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of Th1/Th2-type cytokines. Without Bach2, Treg cells exhibit diminished Foxp3 expression, depleted numbers, hyperactivation, enhanced proliferation, and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim/Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios(-)Foxp3(+) Treg cells and a Treg cell transcriptome skewed toward the Th1/Th2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: 1) is indispensable for TCR/TGF-β-induced Foxp3 expression; and 2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3-driven Th2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal Th2-type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells, and protects against immune-mediated diseases.

Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-β, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.

OBJECTIVE

To investigate the role of immunological parameters in tumorigenesis of cervical cancer in women infected with high risk human papillomavirus (hr-HPV), and determine whether key findings with human material can be recapitulated in the mouse TC1 carcinoma model which expresses hr-HPV epitopes.

METHODS

Epithelial and lymphoid cells in cervical tissues were analyzed by immunohistochemistry and serum IL10 levels were determined by ELISA. Tumor draining lymph nodes were analyzed in the mouse TC1 model by flow cytometry.

RESULTS

The mucosa was infiltrated by CD20+ and CD138+ cells already at cervical intraepithelial neoplasia 1 (CIN1) and infiltration increased in cervical intraepithelial neoplasia 3 (CIN3)/carcinoma in situ (CIS) and invasive cervical cancer (ICC), where it strongly correlated with infiltration by CD32B+ and FoxP3+ lymphocytes. GATA3+ and T-bet+ lymphoid cells were increased in ICC compared to normal, and expression in epithelial cells of the Th2 inflammation-promoting cytokine TSLP and of IDO1 was higher in CIN3/CIS and ICC. As a corollary, serum levels of IL10 were higher in women with CIN3/CIS or ICC than in normals. Finally we demonstrated in the mouse TC1 carcinoma, which expresses hr-HPV epitopes, an increase of cells expressing B cell or plasma cell markers or Fc receptors in tumor-draining than distal lymph nodes or spleen.

CONCLUSIONS

hr-HPV initiates a local Th2 inflammation at an early stage, involving antibody forming cells, and fosters an immunosuppressive microenvironment that aids tumor progression.

While 30%-70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention.

The E2A transcription factors are required for normal T lymphopoiesis and to prevent T-lymphocyte progenitor transformation. Ectopic expression of E2A proteins in E2A-deficient lymphomas results in growth arrest and apoptosis, indicating that these cells remain responsive to the targets of E2A. Here we identify the transcriptional repressor growth factor independent 1B (Gfi1b) as a target of E2A that promotes growth arrest and apoptosis in lymphomas. Gfi1b expression in primary T-lymphocyte progenitors is dependent on E2A and excess Gfi1b prevents the outgrowth of T lymphocyte progenitors in vitro. Gfi1b represses expression of Gata3, a transcription factor whose appropriate regulation is required for survival of lymphomas and T-lymphocyte progenitors. We also show that ectopic expression of Gata3 in lymphomas promotes expression of Gfi1b, indicating that these proteins may function in an autoregulatory loop that maintains appropriate levels of Gata3. Therefore, we propose that E2A proteins prevent lymphoma cell expansion, at least in part through regulation of Gfi1b and modulation of Gata3 expression.

Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision.

Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.

The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

Plant inflorescence meristems and floral meristems possess specific boundary domains that result in proper floral organ separation and specification. HANABA TARANU (HAN) encodes a boundary-expressed GATA3-type transcription factor that regulates shoot meristem organization and flower development in Arabidopsis thaliana, but the underlying mechanism remains unclear. Through time-course microarray analyses following transient overexpression of HAN, we found that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ specification. Transient overexpression of HAN also represses the expression of HAN and three other GATA3 family genes, HANL2 (HAN-LIKE 2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED), and GNL (GNC-LIKE), forming a negative regulatory feedback loop. Genetic analysis indicates that HAN and the three GATA3 family genes coordinately regulate floral development, and their expression patterns are partially overlapping. HAN can homodimerize and heterodimerize with the three proteins encoded by these genes, and HAN directly binds to its own promoter and the GNC promoter in vivo. These findings, along with the fact that constitutive overexpression of HAN produces an even stronger phenotype than the loss-of-function mutation, support the hypothesis that HAN functions as a key repressor that regulates floral development via regulatory networks involving genes in the GATA3 family, along with genes involved in hormone action and floral organ specification.

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.

OBJECTIVE

Previously, we found that gene expression in histologically normal breast epithelium (NlEpi) from women at high breast cancer risk can resemble gene expression in NlEpi from cancer-containing breasts. Therefore, we hypothesized that gene expression characteristic of a cancer subtype might be seen in NlEpi of breasts containing that subtype.

METHODS

We examined gene expression in 46 cases of microdissected NlEpi from untreated women undergoing breast cancer surgery. From 30 age-matched cases [15 estrogen receptor (ER)+, 15 ER-] we used Affymetryix U133A arrays. From 16 independent cases (9 ER+, 7 ER-), we validated selected genes using quantitative real-time PCR (qPCR). We then compared gene expression between NlEpi and invasive breast cancer using four publicly available data sets.

RESULTS

We identified 198 genes that are differentially expressed between NlEpi from breasts with ER+ (NlEpiER+) compared with ER- cancers (NlEpiER-). These include genes characteristic of ER+ and ER- cancers (e.g., ESR1, GATA3, and CX3CL1, FABP7). qPCR validated the microarray results in both the 30 original cases and the 16 independent cases. Gene expression in NlEpiER+ and NlEpiER- resembled gene expression in ER+ and ER- cancers, respectively: 25% to 53% of the genes or probes examined in four external data sets overlapped between NlEpi and the corresponding cancer subtype.

CONCLUSIONS

Gene expression differs in NlEpi of breasts containing ER+ compared with ER- breast cancers. These differences echo differences in ER+ and ER- invasive cancers. NlEpi gene expression may help elucidate subtype-specific risk signatures, identify early genomic events in cancer development, and locate targets for prevention and therapy.

BACKGROUND

National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 suggested the efficacy of adjuvant trastuzumab, even in HER2-negative breast cancer. This finding prompted us to develop a predictive model for degree of benefit from trastuzumab using archived tumor blocks from B-31.

METHODS

Case subjects with tumor blocks were randomly divided into discovery (n = 588) and confirmation cohorts (n = 991). A predictive model was built from the discovery cohort through gene expression profiling of 462 genes with nCounter assay. A predefined cut point for the predictive model was tested in the confirmation cohort. Gene-by-treatment interaction was tested with Cox models, and correlations between variables were assessed with Spearman correlation. Principal component analysis was performed on the final set of selected genes. All statistical tests were two-sided.

RESULTS

Eight predictive genes associated with HER2 (ERBB2, c17orf37, GRB7) or ER (ESR1, NAT1, GATA3, CA12, IGF1R) were selected for model building. Three-dimensional subset treatment effect pattern plot using two principal components of these genes was used to identify a subset with no benefit from trastuzumab, characterized by intermediate-level ERBB2 and high-level ESR1 mRNA expression. In the confirmation set, the predefined cut points for this model classified patients into three subsets with differential benefit from trastuzumab with hazard ratios of 1.58 (95% confidence interval [CI] = 0.67 to 3.69; P = .29; n = 100), 0.60 (95% CI = 0.41 to 0.89; P = .01; n = 449), and 0.28 (95% CI = 0.20 to 0.41; P < .001; n = 442; P(interaction) between the model and trastuzumab < .001).

CONCLUSIONS

We developed a gene expression-based predictive model for degree of benefit from trastuzumab and demonstrated that HER2-negative tumors belong to the moderate benefit group, thus providing justification for testing trastuzumab in HER2-negative patients (NSABP B-47).

The distinctive differentiated states of the CD4+ T helper cells are determined by the set of transcription factors and the genes transcribed by the transcription factors. In vitro induction models, the major determinants of the cytokines present during the T-cell receptor (TCR)-mediated activation process. IL-12 and IFN-γ make Naive CD4+ T cells highly express T-bet and STAT4 and differentiate to TH1 cells, while IL-4 make Naive CD4+ T cells highly express STAT6 and GATA3 and differentiated to TH2 cells. Even through T-bet and GATA3 are master regulators for TH1/TH2 cells differentiation. There are many other transcription factors, such as RUNX family proteins, IRF4, Dec2, Gfi1, Hlx, and JunB that can impair TH1/TH2 cells differentiation. In recent years, noncoding RNAs (microRNA and long noncoding RNA) join in the crowd. The leukocytes should migrate to the right place to show their impact. There are some successful strategies, which are revealed to targeting chemokines and their receptors, that have been developed to treat human immune-related diseases.

GATA transcription factors are important regulators of tissue-specific gene expression during development. GATA2 and GATA3 have been implicated in the regulation of trophoblast-specific genes. However, the regulatory mechanisms of GATA2 expression in trophoblast cells are poorly understood. In this study, we demonstrate that Gata2 is transcriptionally induced during trophoblast giant cell-specific differentiation. Transcriptional induction is associated with displacement of GATA3-dependent nucleoprotein complexes by GATA2-dependent nucleoprotein complexes at two regulatory regions, the -3.9- and +9.5-kb regions, of the mouse Gata2 locus. Analyses with reporter genes showed that, in trophoblast cells, -3.9- and +9.5-kb regions function as transcriptional enhancers in GATA motif independent and dependent fashions, respectively. We also found that knockdown of GATA3 by RNA interference induces GATA2 in undifferentiated trophoblast cells. Interestingly, three other known GATA motif-dependent Gata2 regulatory elements, the -1.8-, -2.8-, and -77-kb regions, which are important to regulate Gata2 in hematopoietic cells are not occupied by GATA factors in trophoblast cells. These elements do not show any enhancer activity and also possess inaccessible chromatin structure in trophoblast cells indicating a context-dependent function. Our results indicate that GATA3 directly represses Gata2 in undifferentiated trophoblast cells, and a switch in chromatin occupancy between GATA3 and GATA2 (GATA3/GATA2 switch) induces transcription during trophoblast differentiation. We predict that this GATA3/GATA2 switch is an important mechanism for the transcriptional regulation of other trophoblast-specific genes.

Hematopoiesis is a classic system with which to study developmental potentials and to investigate gene regulatory networks that control choices among alternate lineages. T-cell progenitors seeding the thymus retain several lineage potentials. The transcription factor PU.1 is involved in the decision to become a T cell or a myeloid cell, and the developmental outcome of expressing PU.1 is dependent on exposure to Notch signaling. PU.1-expressing T-cell progenitors without Notch signaling often adopt a myeloid program, whereas those exposed to Notch signals remain in a T-lineage pathway. Here, we show that Notch signaling does not alter PU.1 transcriptional activity by degradation/alteration of PU.1 protein. Instead, Notch signaling protects against the downregulation of T-cell factors so that a T-cell transcriptional network is maintained. Using an early T-cell line, we describe two branches of this network. The first involves inhibition of E-proteins by PU.1 and the resulting inhibition of Notch signaling target genes. Effects of E-protein inhibition can be reversed by exposure to Notch signaling. The second network is dependent on the ability of PU.1 to inhibit important T-cell transcription factor genes such as Myb, Tcf7 and Gata3 in the absence of Notch signaling. We show that maintenance of Gata3 protein levels by Myb and Notch signaling is linked to the ability to retain T-cell identity in response to PU.1.

IL-4 secreting and nonsecreting cells from Th2 cultures have a similar probability of producing IL-4 upon subsequent stimulation, implying that there is stochastic element in IL-4 production by stimulated Th2 cells. Purified IL-4 producers and nonproducers have similar Gata3 and c-maf mRNA expression. Il4 gene accessibility, analyzed by restriction enzyme accessibility (REA) at sites in the promoter, in the second intron (DNase I hypersensitivity sites HSII and HSIII) and in CNS-1 in the two populations was also similar. However, upon TCR stimulation, site VA, which is 5 kB 3' of exon 4, displayed a striking increase in accessibility but REA was 2- to 3-fold greater in producers than nonproducers. Cyclosporin A treatment inhibited VA opening, implying the involvement of NFAT in increased VA accessibility. Induction of VA accessibility is sensitive to cycloheximide, suggesting an additional factor(s) is needed. Thus, opening of VA is a probabilistic event determining which Th2 cells transcribe Il4.

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.

Serotonin is an important neurotransmitter with multiple functions in the whole central nervous system. Its synthesis, however, is restricted to a very limited number of cells in the brainstem raphe nuclei with a vast axonal network. These cells express markers of the serotonin lineage such as the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 2, the serotonin transporter, and the transcription factor Pet1. Pet1 together with Lmx1b, Nkx2.2, Mash1, Gata2, Gata3, and Phox2b form a transcriptional network, which specifies the differentiation of serotonergic neurons around embryonic day 11 in the mouse. These cells are generated in rhombomeres r1-r3 and r5-r7 caudal to the midbrain- hindbrain organizer under the control of the fibroblast growth factors 4 and 8 and sonic hedgehog (SHH) from precursors, which have produced motoneurons before. Because serotonin is a relevant pathophysiological factor in several neurological diseases such as bipolar disorder and depression tools to generate or maintain serotonergic neurons might be of therapeutic value. Such tools can be assessed in embryonic stem cells, which can be differentiated in vitro to produce serotonergic neurons. Culture systems for these cells including embryoid bodies based and monolayer differentiation have been established, which allows the generation of up to 50% serotonergic neurons in all neurons developed.

BACKGROUND

Immune features of infants with food allergy have not been delineated.

OBJECTIVE

We sought to explore the basic mechanisms responsible for food allergy and identify biomarkers, such as skin prick test (SPT) responses, food-specific IgE levels, and mononuclear cell responses, in a cohort of infants with likely milk/egg allergy at increased risk of peanut allergy.

METHODS

Infants aged 3 to 15 months were enrolled with a positive SPT response to milk or egg and either a corresponding convincing clinical history of allergy to milk or egg or moderate-to-severe atopic dermatitis. Infants with known peanut allergy were excluded.

RESULTS

Overall, 512 infants (67% male) were studied, with 308 (60%) having a history of a clinical reaction. Skin test responses, detectable food-specific IgE, or both revealed sensitization as follows: milk, 78%; egg, 89%; and peanut, 69%. SPT responses and food-specific IgE levels were discrepant for peanut (15% for IgE>> or = 0.35 kU(A)/L and negative SPT response vs 8% for positive SPT response and IgE <0.35 kU(A)/L, P = .001). Mononuclear cell allergen stimulation screening for CD25, cytokine-inducible SH2-containing protein (CISH), forkhead box protein 3 (FOXP3), GATA3, IL10, IL4, IFNG, and T-box transcription factor (TBET) expression by using casein, egg white, and peanut revealed that only allergen-induced IL4 expression was significantly increased in those with clinical allergy to milk (compared with nonallergic subjects) and in those sensitized to peanut, despite the absence of an increase in GATA3 mRNA expression.

CONCLUSIONS

Infants with likely milk/egg allergy are at considerably high risk of having increased peanut-specific IgE levels (potential allergy). Peanut-specific serum IgE levels were a more sensitive indicator of sensitization than SPT responses. Allergen-specific IL4 expression might be a marker of allergic risk. Absence of an increase in GATA3 mRNA expression suggests that allergen-specific IL-4 might not be of T-cell origin.