Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

The biological role of Langerin(+) dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin(+) DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

Altered interactions within FY/AtCPSF complexes required for Arabidopsis FCA-mediated chromatin silencing.

The role of RNA metabolism in chromatin silencing is now widely recognized. We have studied the Arabidopsis RNA-binding protein FCA that down-regulates an endogenous floral repressor gene through a chromatin mechanism involving histone demethylase activity. This mechanism needs FCA to interact with an RNA 3' processing/polyadenylation factor (FY/Pfs2p), but the subsequent events leading to chromatin changes are unknown. Here, we show that this FCA-FY interaction is required for general chromatin silencing roles where hairpin transgenes induce DNA methylation of an endogenous gene. We also show 2 conserved RNA processing factors, AtCPSF100 and AtCPSF160, but not FCA, are stably associated with FY in vivo and form a range of different-sized complexes. A hypomorphic fy allele producing a shorter protein, able to provide some FY functions but unable to interact with FCA, reduces abundance of some of the larger MW complexes. Suppressor mutants, which specifically disrupt the FY motif through which FCA interacts, also lacked these larger complexes. Our data support a model whereby FCA, perhaps after recognition of a specific RNA feature, transiently interacts with FY, an integral component of the canonical RNA 3' processing machinery, changing the interactions of the different RNA processing components. These altered interactions would appear to be a necessary step in this RNA-mediated chromatin silencing.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-gamma can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-gamma secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-gamma responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-gamma secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-gamma secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-gamma that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

Adiponectin is produced by lymphocytes and is a negative regulator of granulopoiesis

Lymphocytes have long been established to play an important role in the regulation of hematopoiesis and produce many cytokines that act on hematopoietic progenitor cells. Previous studies by our group have shown that normal, unstimulated lymphocytes produce a protein that inhibits normal bone marrow GM colony formation. Adiponectin is an adipokine that has been demonstrated to act as a negative regulator of hematopoiesis and immune function. This study aimed to determine if the inhibitory molecule that we described previously was adiponectin. Here, we show transcription, translation, and secretion of adiponectin from lymphocytes and demonstrate that its receptors, AdipoR1 and AdipoR2, are expressed by bone marrow MNCs. We show that although the adiponectin expression is low in lymphocytes, it is sufficient to induce a significant inhibitory effect on GM precursors (CFU-GM) and activate the AMPK pathway in these cells. The regulation of adiponectin production by lymphocytes and its detailed function in suppressing GM colony formation need to be elucidated now. Our findings suggest a functional role for adiponectin as a negative regulator of granulopoiesis. J. Leukoc. Biol. 88: 807-811; 2010.

Methods for measuring proteasome activity: Current limitations and future developments

The proteasome has been validated as a therapeutic target, with proteasome inhibitors showing particular efficacy in the treatment of Multiple Myeloma. A wide range of methods have been developed to profile proteasome activity. These include the current method of choice fluorogenic peptide substrates, as well as bioluminescent imaging, immunological methods, and more recently, site-specific fluorescent probes. The aim of this review is to evaluate the currently available methods for profiling proteasome activity and their suitability for use in translational studies. Ongoing development of techniques for profiling proteasome activity will facilitate future research into proteasome-related pathologies, thus accelerating the development of more specific drug regimes. (C) 2010 Elsevier Ltd. All rights reserved.

Bortezomib induces apoptosis in primitive chronic myeloid leukemia cells including LTC-IC and NOD/SCID repopulating cells

Chronic myeloid leukemia (CML) is treated effectively with tyrosine kinase inhibitors (TKIs); however, 2 key problems remain-the insensitivity of CML stem and progenitor cells to TKIs and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and proteasome inhibitors (PIs) are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in chronic phase (CP) CD34(+) CML cells at clinically achievable concentrations. We also show that bortezomib targets primitive CML cells, with effects on CD34(+)38(-), long-term culture-initiating (LTC-IC) and nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells. Bortezomib is not selective for CML cells and induces apoptosis in normal CD34(+)38(-) cells. The effects against CML cells are seen when bortezomib is used alone and in combination with dasatinib. Bortezomib causes proteasome but not BCR-ABL inhibition and is also effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. By targeting both TKI-insensitive stem and progenitor cells and TKI-resistant BCR-ABL mutations, we believe that bortezomib offers a potential therapeutic option in CML. Because of known toxicities, including myelosuppression, the likely initial clinical application of bortezomib in CML would be in resistant and advanced disease. (Blood. 2010;115:2241-2250)