The thymus is regarded as the primary site for T-cell lymphopoiesis, but very little is known about the lineage inter-relationships of cells within that organ. At least four subpopulations of mouse thymocytes can be defined on the basis of staining with monoclonal antibodies directed against the T-cell differentiation antigens Lyt-2 and L3T4 (ref. 2). Thus immunocompetent (medullary) thymocytes, like peripheral T cells, express either Lyt-2 (cytotoxic phenotype) or L3T4 (helper phenotype) but not both, whereas non-functional (cortical) thymocytes express both markers. In addition, a small subpopulation comprising 2-3% of cells in the thymus and expressing neither Lyt-2 nor L3T4 has recently been described. The latter cells have the properties of intrathymic 'stem cells' in that they are the first to appear in the embryonic thymus and at least some can be shown to give rise, both in vivo (ref. 4. and our unpublished data) and in vitro, to other thymocyte subpopulations. We show here that 50% of Lyt-2-/L3T4- cells in the adult thymus express receptors for the polypeptide growth hormone interleukin-2 (IL-2) whereas other cells in the thymus do not. Furthermore, immunohistochemical localization studies on frozen sections indicate a disperse distribution of IL-2 receptor-positive cells in both the cortex and medulla. These novel findings have potential implications in the context of current models of differentiation pathways within the thymus.

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In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I–like function may contribute to the chicken’s susceptibility to highly pathogenic influenza.

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Interleukin-2 (IL-2) is a T-cell-derived polypeptide hormone of 133 amino acids which exerts its growth-promoting activity via a surface receptor. Originally, IL-2 was believed to be a unique growth factor for activated T cells; more recent studies, however, have demonstrated that certain B-cell tumours as well as normal activated B lymphocytes express a surface molecule which is recognized by monoclonal antibodies directed against the IL-2 receptor. Furthermore, we and others have shown recently that activated B cells proliferate in response to either immunoaffinity-purified or recombinant IL-2. These controversial findings prompted us to undertake a detailed quantitative comparison of IL-2 receptor expression on activated B and T cells. We show here, using biosynthetically labelled IL-2(3H-IL-2) and anti-IL-2 receptor antibody (3H-PC61) that activated B and T cells express both high-affinity (apparent dissociation constant, Kd approximately 20 pM) and low-affinity (Kd approximately 1,000 pM) IL-2 receptors. Binding of IL-2 to both classes of receptor is inhibited by the monoclonal anti-IL-2 receptor antibody PC61. B blasts express half as many total IL-2 binding sites or PC61 binding sites as T blasts, and the ratio of the number of low- to high-affinity receptors for each cell type is approximately 10:1. Immunoprecipitation analysis of surface-labelled blasts indicates that B and T cells have IL-2 receptors of similar relative molecular mass. Taken together, these data suggest strongly that IL-2 can act as a growth hormone for both B and T lymphocytes.

We have investigated the biochemical basis for the activation of interleukin 2 receptor a-subunit gene expression in primary human T lymphocytes by a cytokine (tumor necrosis factor a), a T-cell mitogen (phorbol 12-myristate 13-acetate), and the transactivator protein (Tax) from the type I human T-cell leukemia virus. Using in vivo transfection techniques specificially designed for these primary T cells in coajunction with in vitro gel retardation and DNA footprinting assays, we found that activation of the IL-2Ra promoter by each of these agents involves the induction of nuclear proteins that specifically interact with a #cB-like enhancer element (i.e., an element resembling the immunoglobulin K-chain enhancer sequence recognized by transcription factor NF-iB). DNA-protein crosslinking studies revealed that primary T cells express at least three different inducible DNA-binding proteins (50)(51)(52)(53)(54)(55)(70)(71)(72)(73)(74)(75)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90) that specifically interact with this IL-2Ra cB element.The proliferation of human T lymphocytes involves the coordinated activation of genes encoding the T-cell growth factor interleukin 2 (IL-2) and its membrane receptor (IL-2R) (1, 2). The functional high-affinity IL-2R complex consists of at least two distinct IL-2-binding protein subunits, IL-2Ra (Tac, p55, CD25) (3) and IL-2R1 (p70-75) (4-7). Both ofthese receptor subunits appear to be required for effective growthsignal transduction in the presence of physiological concentrations of 9). Since resting T cells constitutively express IL-2R3 (7), the induction of IL-2Ra gene expression is important for the regulation of high-affinity IL-2R display and T-cell proliferation. Cell surface expression of IL-2Ra can be induced by a variety of stimuli, including antigens, the transactivator protein (Tax) of type I human T-cell leukemia virus (HTLV-I) (10), and nonspecific mitogens such as phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) (11). In addition, tumor necrosis factor a (TNF-a), a macrophage-and T-cell-derived cytokine that displays a wide spectrum of biological activities (12), has been shown to induce IL-2Ra gene expression in both normal and leukemic T cells (13,14).Previous studies of the regulation of IL-2Ra gene activation have used long-term cultured leukemic T-cell lines as approximate experimental models for activation events occurring in primary T cells (15)(16)(17)(18)(19)(20)(21). Studies of one such T-cell line, Jurkat, suggested that the interaction of inducible DNA-binding proteins with a KB-like promoter element is important in the Tax activation of the IL-2Ra gene (19-21); however, disparate requirements for this element have been described with phorbol ester as the inducer (18-21).In the studies reported here, we analyzed the cis-acting regulatory sequences and trans-acting factors operational in the activation of IL-2Ra gene expression in primary human T cells induced with TNF-a, PMA, or Tax. Functional transfection studies employing a ...

We have cloned the gene for chicken interferon-gamma (ChIFN-gamma) from a cDNA expression library generated from a T cell line (CC8.1h) that produces high levels of IFN-gamma activity. CC8.1h constitutively produces IFN activity that shares physiochemical properties with mammalian IFN-gamma. ChIFN-gamma, when secreted by CC8.1h or expressed in transfected COS cells, is heat labile, inactivated by exposure to pH 2, and capable of inducing nitrite production by chicken macrophages. These properties clearly distinguish it from chicken and mammalian type I IFN. The ChIFN-gamma gene codes for a predicted mature protein of 145 amino acids with a molecular mass of 16.8 kD. There are two potential N-glycosylation sites located near the N terminus. ChIFN-gamma protein shares significant amino acid homology with mammalian IFN-gamma proteins; in particular it also contains the highly conserved motifs that are present in all mammalian IFN-gamma proteins. ChIFN-gamma is 35 and 32% identical to the equine and human counterparts, respectively, but shares only 15% homology with chicken type I IFN. These findings show that the emergence of the two principal types of IFN predates the divergence of avians and mammals that occurred some 350 million years ago.

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.