Control of Common Bunt in Organic Wheat.

Common bunt, caused by the seedborne and soilborne pathogens Tilletia caries and T. laevis, has re-emerged as a major disease in organic wheat. In conventional agriculture, common bunt is routinely managed with the use of synthetic chemical seed treatments. For this reason, common bunt is a relatively unimportant disease in conventional agriculture. However, since synthetic chemical inputs are prohibited in organic agriculture, common bunt is a major threat once more in organic wheat and seed production. The challenge today is to manage the disease without the use of chemical seed treatments. This review reports on the management of common bunt under organic farming systems, mainly through host resistance and organic seed treatments. We report the history of screening wheat germplasm for bunt resistance, the search for new sources of resistance, and identification and mapping of bunt resistance genes. Since the pathogen has a gene-for-gene relationship with the host, this review also includes a summary of work on pathogen race identification and virulence patterns of field isolates. Also included are studies on the physiological and molecular basis of host resistance. Alternative seed treatments are discussed, including physical seed treatments, and microbial-based and plant-based treatments acceptable in organic systems. The article concludes with a brief discussion on the current gaps in research on the management of common bunt in organic wheat.

Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells.

CD4(+) and CD8(+) T cells exhibit important differences in their major effector functions. CD8(+) T cells provide protection against pathogens through cytolytic activity, whereas CD4(+) T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-gamma, which can contribute to protective immunity. Here we show that CD4(+) and CD8(+) T cells differ in their regulation of IFN-gamma production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-gamma induced by interleukin (IL)-12/IL-18 signaling, but only CD4(+) T cells require Stat4 for IFN-gamma induction via the TCR pathway. In response to antigen, CD8(+) T cells can produce IFN-gamma independently of IL-12, whereas CD4(+) T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-gamma production based on differences in TCR signaling between CD4(+) and CD8(+) T cells.

Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells.

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

T cell genetic background determines default T helper phenotype development in vitro.

A host's ability to resist certain pathogens such as Leishmania major can depend upon the phenotype of T helper (Th) subset that develops. Different murine genetic backgrounds are known to significantly alter the direction of Th subset development, although the cellular basis of this influence is poorly understood. To examine the basis of this effect we used an in vitro alpha/beta-T cell receptor (TCR) transgenic system for analysis of Th phenotype development. To control for TCR usage, we derived the DO11.10 alpha/beta-TCR transgene in several genetic backgrounds. Our findings suggest that the effects of genetic background on Th phenotype development reside within the T cell, and not the antigen-presenting cell compartment. Transgenic T cells from both the B10.D2 and BALB/c backgrounds showed development toward either the Th1 or Th2 phenotype under the strong directing influence of interleukin (IL) 12 and IL4, respectively. However, when T cells were activated in vitro under neutral conditions in which exogenous cytokines were not added, B10.D2-derived T cells acquired a significantly stronger Th1 phenotype than T cells from the BALB/c background, correspondent with in vivo Th responses to Leishmania in these strains. Importantly, these cytokine differences resulted in distinct functional properties, because B10.D2- but not BALB/c-derived T cells could induce macrophage production of nitric oxide, an important antimicrobial factor. Thus, the genetically determined default Th phenotype development observed in vitro may correspond to in vivo Th subset responses for pathogens such as Leishmania which do not initiate strong Th phenotype-directing signals.

The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model.

The dose of foreign antigen can influence whether a cell-mediated or humoral class of immune response is elicited, and this may be largely accounted for by the development of CD4+ T helper cells (Th) producing distinct sets of cytokines. The ability of antigen dose to direct the development of a Th1 or Th2 phenotype from naive CD4+ T cells, however, has not been demonstrated. In this report, we show that the antigen dose used in primary cultures could directly affect Th phenotype development from naive DO11.10 TCR-alpha beta-transgenic CD4+ T cells when dendritic cells or activated B cells were used as the antigen-presenting cells. Consistent with our previous findings, midrange peptide doses (0.3-0.6 microM) directed the development of Th0/Th1-like cells, which produced moderate amounts of interferon gamma (IFN-gamma). As the peptide dose was increased, development of Th1-like cells producing increased amounts of IFN-gamma was initially observed. At very high (> 10 microM) and very low (< 0.05 microM) doses of antigenic peptide, however, a dramatic switch to development of Th2-like cells that produced increasing amounts of interleukin 4 (IL-4) and diminishing levels of IFN-gamma was observed. This was true even when highly purified naive, high buoyant density CD4+ LECAM-1hi T cells were used, ruling out a possible contribution from contaminating "memory" phenotype CD4+ T cells. Neutralizing anti-IL-4 antibodies completely inhibited the development of this Th2-like phenotype at both high and low antigen doses, demonstrating a requirement for endogenous IL-4. Our findings suggest that the antigen dose may affect the levels of endogenous cytokines such as IL-4 in primary cultures, resulting in the development of distinct Th cell phenotypes.

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Friend of GATA-1 represses GATA-3-dependent activity in CD4+ T cells.

The development of naive CD4+ T cells into a T helper (Th) 2 subset capable of producing interleukin (IL)-4, IL-5, and IL-13 involves a signal transducer and activator of transcription (Stat)6-dependent induction of GATA-3 expression, followed by Stat6-independent GATA-3 autoactivation. The friend of GATA (FOG)-1 protein regulates GATA transcription factor activity in several stages of hematopoietic development including erythrocyte and megakaryocyte differentiation, but whether FOG-1 regulates GATA-3 in T cells is uncertain. We show that FOG-1 can repress GATA-3-dependent activation of the IL-5 promoter in T cells. Also, FOG-1 overexpression during primary activation of naive T cells inhibited Th2 development in CD4+ T cells. FOG-1 fully repressed GATA-3-dependent Th2 development and GATA-3 autoactivation, but not Stat6-dependent induction of GATA-3. FOG-1 overexpression repressed development of Th2 cells from naive T cells, but did not reverse the phenotype of fully committed Th2 cells. Thus, FOG-1 may be one factor capable of regulating the Th2 development.

Heterogeneity of single cell cytokine gene expression in clonal T cell populations.

T helper type 0 (Th0), Th1, and Th2 CD4+ T cell clones derived from a T cell receptor alpha/beta (TCR-alpha/beta) transgenic mouse were activated by antigen presented on "artificial" antigen-presenting cells that expressed or lacked the costimulatory molecule B7-1, and were analyzed for single cell cytokine mRNA expression by in situ hybridization. There was significant heterogeneity in the frequency of T cells that expressed individual cytokine mRNAs within each clonal population, suggesting that transcriptional control of each of the cytokine genes was not coordinate within an individual cell. The majority of antigen-stimulated Th1 cells expressed mRNA for interferon gamma (IFN-gamma), but far fewer cells in the same population expressed interleukin 2 (IL-2). Similarly, the frequency of IL-4-expressing cells was greater than that of IL-5- or IL-10-expressing cells in the same Th2 population, but the difference in expression frequencies was more variable between clones. The expression frequencies of each of the cytokines was quite heterogeneous in the antigen-activated Th0 population. The principal effect of increased antigen on the activation of individual cytokine genes in each of the clonal populations was to increase recruitment of mRNA-positive cells, with little or no effect on the level of cytokine mRNA expression in individual positive cells. The effects of B7 costimulation were variable depending on the cytokine gene analyzed. B7 costimulation markedly increased the frequency and the level of IL-2 mRNA expression in individual positive cells in the Th1 and Th0 populations, with less effect on the recruitment and single cell expression level of IFN-gamma. IL-4 frequencies were modestly increased by B7 costimulation of the Th2 clones, but there was no detectable increase in single cell IL-4 expression level. The observed patterns of cytokine mRNA expression favor a model of T cell activation in which all-or-none, rather than graded, responses of cytokine genes are dominant.

Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.

In order to examine the mechanisms by which clonal deletion of autoreactive T cells occurs, a peptide antigen was used to induce deletion of antigen-reactive thymocytes in vivo. Mice transgenic for a T cell receptor (TCR) that reacts to this peptide contain thymocytes that progress from the immature to the mature phenotype. Intraperitoneal administration of the peptide antigen to transgenic mice results in a rapid deletion of the immature CD4+ CD8+ TCRlo thymocytes. Apoptosis of cortical thymocytes can be seen within 20 hours of treatment. These results provide direct evidence for the in vivo role of apoptosis in the development of antigen-induced tolerance.

Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages.

Development of the appropriate CD4+ T helper (TH) subset during an immune response is important for disease resolution. With the use of naïve, ovalbumin-specific alpha beta T cell receptor transgenic T cell, it was found that heat-killed Listeria monocytogenes induced TH1 development in vitro through macrophage production of interleukin-12 (IL-12). Moreover, inhibition of macrophage production of IL-12 may explain the ability of IL-10 to suppress TH1 development. Murine immune responses to L. monocytogenes in vivo are of the appropriate TH1 phenotype. Therefore, this regulatory pathway may have evolved to enable innate immune cells, through interactions with microbial pathogens, to direct development of specific immunity toward the appropriate TH phenotype.

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Ligand-induced autoregulation of IFN-gamma receptor beta chain expression in T helper cell subsets.

Interferon gamma (IFN-gamma) responsiveness in certain cells depends on the state of cellular differentiation or activation. Here an in vitro developmental system was used to show that IFN-gamma produced during generation of the CD4+ T helper cell type 1 (TH1) subset extinguishes expression of the IFN-gamma receptor beta subunit, resulting in TH1 cells that are unresponsive to IFN-gamma. This beta chain loss also occurred in IFN-gamma-treated TH2 cells and thus represents a specific response of CD4+ T cells to IFN-gamma rather than a TH1-specific differentiation event. These results define a mechanism of cellular desensitization where a cytokine down-regulates expression of a receptor subunit required primarily for signaling and not ligand binding.

Phorbol ester receptors: autoradiographic identification in the developing rat.

Autoradiography with 3H-labeled phorbol dibutyrate was used for the light microscopic detection of phorbol ester receptors in rat fetuses. In 15- and 18-day fetuses, as well as in adult rats, receptors were found to be concentrated in the central nervous system. The localization of receptors in the ventral marginal zone of the fetal neural tube, the lens of the eye, and other sites suggests a role for phorbol ester receptors in cellular process extension and cell-cell interaction.

Genetic susceptibility to Leishmania: IL-12 responsiveness in TH1 cell development.

The genetic background of T lymphocytes influences development of the T helper (TH) phenotype, resulting in either resistance or susceptibility of certain mouse strains to pathogens such as Leishmania major. With an in vitro model system, a difference in maintenance of responsiveness of T cells to interleukin-12 (IL-12) was detected between BALB/c and B10.D2 mice. Although naive T cells from both strains initially responded to IL-12, BALB/c T cells lost IL-12 responsiveness after stimulation with antigen in vitro, even when cocultured with B10.D2 T cells. Thus, susceptibility of BALB/c mice to infection with L. major may derive from the loss of the ability to generate IL-12-induced TH1 responses rather than from an IL-4-induced TH2 response.

ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells.

During normal tumor growth and in response to some therapies, tumor cells experience acute or chronic deprivation of nutrients and oxygen and induce tumor vascularization. While this occurs predominately through sprouting angiogenesis, tumor cells have also been shown to directly contribute to vessel formation through vascular mimicry (VM) and/or endothelial transdifferentiation. The extrinsic and intrinsic mechanisms underlying tumor cell adoption of endothelial phenotypes, however, are not well understood. Here we show that serum withdrawal induces mesenchymal breast cancer cells to undergo VM and that knockdown of the epithelial-to-mesenchymal transition (EMT) regulator, Zinc finger E-box binding homeobox 1 (ZEB1), or overexpression of the ZEB1-repressed microRNAs (miRNAs), miR-200c, miR-183, miR-96 and miR-182 inhibits this process. We find that secreted proteins Fibronectin 1 (FN1) and serine protease inhibitor (serpin) family E member 2 (SERPINE2) are essential for VM in this system. These secreted factors are upregulated in mesenchymal cells in response to serum withdrawal, and overexpression of VM-inhibiting miRNAs abrogates this upregulation. Intriguingly, the receptors for these secreted proteins, low-density lipoprotein receptor-related protein 1 (LRP1) and Integrin beta 1 (ITGB1), are also targets of the VM-inhibiting miRNAs, suggesting that autocrine signaling stimulating VM is regulated by ZEB1-repressed miRNA clusters. Together, these data provide mechanistic insight into the regulation of VM and suggest that miRNAs repressed during EMT, in addition to suppressing migratory and stem-like properties of tumor cells, also inhibit endothelial phenotypes of breast cancer cells adopted in response to a nutrient-deficient microenvironment.

Role of MHC and antigens in T-cell development.

The role of self-peptides in influencing the development of the T-cell repertoire has been the focus of recent studies. The findings suggest that the recognition of self-peptides bound to MHC proteins in the thymus is part of the thymic self-recognition process that results in selective maturation, or positive selection of T cells.

T lymphocyte differentiation in the periphery.

The development of immune responses is significantly influenced by emerging patterns of cytokine expression in activated CD4+ T cells. Recent efforts have clarified both cellular and molecular mechanisms, and within the past year include significant observations on potential sources of IL-4 leading to Th2 development against certain pathogens, and insights into early responses and genetic susceptibility to experimental murine Leishmaniasis and transcriptional regulation of the IL-4 locus. Advances in Th1 development have included greater understanding of IL-12 receptors in Th1 development, data regarding IFN-gamma gene expression and clarification of the action of the new cytokine IL-18.

Stability and commitment in T helper cell development.

Specialized effector activities that are required to eliminate various pathogens involve cytokines produced by specialized CD4(+) T cells subsets, dogmatically termed Th1 and Th2 cells. Despite some oversimplifications, this paradigm is useful for organizing the complex pathways that control forward and backward movements along the road of T cell differentiation. Effective immune memory relies, in part, on the maintenance of the T helper phenotype. This review will address basic issues that relate to the maintenance or reversibility of Th1/Th2 states within the CD4(+) T cell lineage.

Structure and specificity of GATA proteins in Th2 development.

Development of Th2 subset of CD4+ T cells involves the interleukin-4 (IL-4)- and Stat6-dependent increase in GATA-3 expression during primary activation. Recently we reported that the phenotypic stability and factor independence of Th2 cells involves acquisition of an intracellular pathway that maintains GATA-3 expression. Evidence from retroviral expression studies implied that this pathway involved an autoactivation of GATA-3 expression, since Stat6-deficient T cells induced endogenous GATA-3 when infected with GATA-3-expressing retroviruses. That study left unresolved the issue of whether GATA-3 autoactivation was direct or indirect. Several other Th2-specific transcription factors have been described, including c-Maf and JunB. We therefore examined the ability of these other transcription factors to induce GATA-3 expression and promote Th2 development. Neither c-Maf nor JunB induced Th2 development in Stat6-deficient CD4+ T cells, in contrast to GATA-3. Consistent with this indication of a possible direct autoactivation pathway, we also observed that heterologous GATA family proteins GATA-1, GATA-2, and GATA-4 were also capable of inducing GATA-3 expression in developing Stat6-deficient T cells and promote Th2 development. Mutational analysis revealed evidence for two distinct mechanisms of GATA-3 action. IL-4 induction by GATA-3 required each of the functional domains to be present, whereas repression of gamma interferon could occur even when mutants of GATA-3 lacking the second transactivation domain, TA2, were expressed. The GATA-dependent induction of the GATA-3 but not the other GATA genes in T cells suggests that T-cell-specific cis elements within the GATA-3 locus likely cooperate with a general GATA recognition motif to allow GATA-3-dependent autoactivation.