A threshold level of NFATc1 activity facilitates thymocyte differentiation and opposes notch-driven leukaemia development.

International audienceNFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1beta expression, preTCR-positive thymocytes express both Nfatc1beta and P1 promoter-derived Nfatc1alpha transcripts. Inducing NFATc1alpha activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1beta from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action

Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications

<p>Abstract</p> <p>Background</p> <p><it>Acidithiobacillus ferrooxidans </it>is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, γ-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth. It thrives at extremely low pH (pH 1–2) and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. The lack of a well-developed system for genetic manipulation has prevented thorough exploration of its physiology. Also, confusion has been caused by prior metabolic models constructed based upon the examination of multiple, and sometimes distantly related, strains of the microorganism.</p> <p>Results</p> <p>The genome of the type strain <it>A. ferrooxidans </it>ATCC 23270 was sequenced and annotated to identify general features and provide a framework for <it>in silico </it>metabolic reconstruction. Earlier models of iron and sulfur oxidation, biofilm formation, quorum sensing, inorganic ion uptake, and amino acid metabolism are confirmed and extended. Initial models are presented for central carbon metabolism, anaerobic metabolism (including sulfur reduction, hydrogen metabolism and nitrogen fixation), stress responses, DNA repair, and metal and toxic compound fluxes.</p> <p>Conclusion</p> <p>Bioinformatics analysis provides a valuable platform for gene discovery and functional prediction that helps explain the activity of <it>A. ferrooxidans </it>in industrial bioleaching and its role as a primary producer in acidic environments. An analysis of the genome of the type strain provides a coherent view of its gene content and metabolic potential.</p

Characterization of monocyte-activating tumour cell membrane structures

Tumor cells are known to activate monocytes/macrophages and it has been shown that this stimulation was conferred by tumour-cell membranes. In order to analyse the relevant structures for tumor cell-specific TNF-induction monocytes from healthy donors were cultured in the presence of plasma membrane preparations from Jurkat or K562 cells. Both tumour cell lines revealed a monocyte-stimulating plasma membrane component of about 45 kDa. The TNF-inducing factor exhibited characteristics of a glycoprotein with the carbohydrate moiety as the structure responsible for stimulation. CD2, a glycosylated T-cell specific membrane component, was identified as being involved in monocyte activation in the case of the Jurkat cells whereas the identity of the activating structure on K562 cells is still unknown. From the data presented here indicating the importance of carbohydrate structures for monocyte activation we conclude that altered glycosylation of cell surface molecules of tumour cells might be responsible for tumour cell-induced monocyte stimulation

Adhesion molecules on freshly recovered T leukemias promote tumor- directed lympholysis

Abstract Besides facilitating cell to cell adhesion, the molecular interactions between CD2 and its ligand CD58 (lymphocyte function-associated antigen- 3 [LFA-3]), as well as between CD11a/18 (LFA-1) and CD54 (intercellular adhesion molecule-1) have recently been recognized to participate in lymphocyte activation, recirculation, and effector function, including cytolytic activity towards tumor cells. We have investigated the role of CD2/CD58 and CD11a/18/CD54 interactions in cellular immune responses directed towards freshly recovered human T-cell leukemias. The data support the notion that downregulation of CD54 and CD58 correlates with enhanced numbers of blasts in circulation and unsusceptibility to killing by autologous cytotoxic lymphocytes. Importantly, after induction of CD54 and CD58 expression on leukemic cells by recombinant cytokines such as tumor necrosis factor-alpha, tumor cells become highly susceptible to lymphocyte-mediated lysis in vitro. Our findings, therefore, stress the point that successful immunotherapy of malignant disease may be facilitated by influencing not only the immune response itself, but also adhesion molecules on the malignant tumor targets.</jats:p

Adhesion molecules on freshly recovered T leukemias promote tumor- directed lympholysis

Besides facilitating cell to cell adhesion, the molecular interactions between CD2 and its ligand CD58 (lymphocyte function-associated antigen- 3 [LFA-3]), as well as between CD11a/18 (LFA-1) and CD54 (intercellular adhesion molecule-1) have recently been recognized to participate in lymphocyte activation, recirculation, and effector function, including cytolytic activity towards tumor cells. We have investigated the role of CD2/CD58 and CD11a/18/CD54 interactions in cellular immune responses directed towards freshly recovered human T-cell leukemias. The data support the notion that downregulation of CD54 and CD58 correlates with enhanced numbers of blasts in circulation and unsusceptibility to killing by autologous cytotoxic lymphocytes. Importantly, after induction of CD54 and CD58 expression on leukemic cells by recombinant cytokines such as tumor necrosis factor-alpha, tumor cells become highly susceptible to lymphocyte-mediated lysis in vitro. Our findings, therefore, stress the point that successful immunotherapy of malignant disease may be facilitated by influencing not only the immune response itself, but also adhesion molecules on the malignant tumor targets.</jats:p

Purified lymphocyte function-associated antigen-3 and T11 target structure are active in CD2-mediated T cell stimulation.

In this study we have used cells expressing LFA-3 or T11TS, the human and sheep forms of the ligand of CD2, as well as the purified LFA-3 and T11TS molecules themselves to study their effects on T cell activation via the CD2-mediated "alternative pathway". Sheep red blood cells, which bind to CD2 via T11TS in E-rosette formation, and human autologous monocytes, which express the LFA-3 molecule, both induce proliferation of resting T cells in the presence of per se submitogenic concentrations of anti-T11(2) plus anti-T11(3) monoclonal antibodies (mAb). This effect is blocked by mAb to LFA-3, T11TS and CD2 known to inhibit CD2-ligand interaction. In addition, purified LFA-3 and T11TS, when added at ng amounts to cultures containing submitogenic concentrations of anti-T11(2 + 3) mAb, are also strongly mitogenic for resting human T cells. Thus, both LFA-3 and T11TS are potent co-stimulators of the alternative pathway of T cell activation but by themselves do not provide a mitogenic signal. This finding is discussed with regard to a physiological role of CD2-LFA-3 interaction in T cell activation