IntroductionIndoleamine 2,3-dioxygenase (IDO) is a key enzyme in the tryptophan metabolism that catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway. 1 Tryptophan starvation by IDO consumption inhibits T-cell activation, 1,2 while products of tryptophan catabolism, such as kynurenine derivatives and O 2 -free radicals, regulate T-cell proliferation and survival. 1,3 Thus, IDO has been shown to exert an immunosuppressive activity, and cell populations, including regulatory dendritic cells (DCs) and bone marrow (BM)-derived mesenchymal stem cells (MSCs), expressing IDO have the capacity to suppress T-cell responses to auto-and alloantigens. 4,5 A wide variety of human solid tumors express IDO. 6 More recently, we demonstrated that also acute myeloid leukemia (AML) cells, but not their normal counterparts (ie, CD34 ϩ hematopoietic stem/progenitor cells [HSCs]), express an active IDO protein, which converts tryptophan into kynurenine and inhibits allogeneic T-cell proliferation. 7 Naturally arising CD4 ϩ CD25 ϩ Foxp3 ϩ T regulatory (T reg ) cells are known to suppress most types of immune response, 8,9 including antitumor immunity. [10][11][12][13] IDO is expressed and is functionally active in placenta, which, in turn, is infiltrated by CD4 ϩ CD25 ϩ T reg cells. [14][15][16] Moreover, Candida albicans infection increases the number of T reg cells because of IDO induction in host antigenpresenting cells (APCs). 17 In human cancers, tumor-draining lymph nodes contain IDO-expressing DCs that enhance T reg cell function. 18 These data suggest the close relationship between IDO activity and the occurrence of T reg cells, 19 but the mechanism governing the generation of T reg cells by IDO-expressing tumors is presently unknown.In the present study, we investigated whether the expression of IDO by AML cells may play a direct role in the development of T reg cells. Materials and methods CellsAll human samples were obtained after informed consent was signed, according to institutional guidelines. Approval was obtained from Bologna Hospital Ethical Committee. Buffy coats were obtained from healthy adults during the preparation of transfusion products. BM and/or peripheral blood (PB) samples including at least 70% leukemic cells were harvested from 76 patients with AML at diagnosis. CD3 ϩ and CD4 ϩ cells were purified from the mononuclear cell (MNC) fraction by MiniMacs high-gradient magnetic separation column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions (purity of CD3 ϩ and CD4 ϩ cell populations was always greater than 95%). MSCs were generated from BM cells as previously reported. 20 Murine A20 and CT26 cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) or MEM (Whittaker Bioproducts) supplemented with 10% FCS (Sera Lab, Crawley Down, United The online version of this manuscript contains a data supplement.The publication costs of ...

IntroductionDuring the last few years the biologic activity of extracellular nucleotides has been the focus of increasing attention. Responses to extracellular adenosine triphosphate (ATP) or uridine triphosphate (UTP) as different as cell proliferation, differentiation, chemotaxis, cytokine secretion, release of lysosomal constituents, generation of reactive oxygen or nitrogen species, and cell death have been reported by different groups. [1][2][3][4][5][6][7][8][9] The effects of extracellular nucleotides are mediated by specific plasma membrane receptors, which are classified into 2 families: metabotropic P2Y receptors (P2YRs) 10 and ionotropic P2X receptors (P2XRs). [11][12][13] P2YRs are typical G protein-coupled receptors with 7 transmembrane domains. Eight P2YRs have been cloned so far. Signal transduction occurs via activation of phospholipase C or stimulation/ inhibition of adenylate cyclase. ATP is an agonist at all P2Y subtypes, with the exception of P2Y 12 and P2Y 14 , where adenosine diphosphate (ADP) and uridine diphosphate (UDP)-glucose are the preferred agonists, respectively, and ATP is either ineffective or an antagonist. UTP is a better agonist than ATP at P2Y 4 and P2Y 6 , whereas at P2Y 2 ATP and UTP are equipotent. P2XRs are multimeric plasma membrane ion channels directly gated by ATP that mediate fast permeability changes to monovalent and divalent cations (Na ϩ , K ϩ , and Ca 2ϩ ). Seven P2X subunits have been cloned so far. P2Rs are widely expressed on mature blood cells. [14][15][16][17][18][19][20] Moreover, the expression and function of the P2X 7 R on B-cell chronic lymphocytic leukemia (B-CLL) cells correlate with the severity of the disease, and targeting of such receptors has been proposed as a novel form of treatment for chronic leukemia. 21 No information is currently available on the expression and function of P2Rs on human hematopoietic stem cells (HSCs). HSCs are identified by the expression of the CD34 antigen, a cell membrane phosphoglycoprotein present on human bone marrow (BM), peripheral blood (PB), and cord blood progenitors. 22 Animal models 23 and clinical transplantation studies 24 using highly enriched cell populations have demonstrated the capacity of CD34 ϩ cells to behave as true stem cells because they engraft lethally irradiated allogeneic hosts. Moreover, retrovirally marked autologous CD34 ϩ grafts sustain long-term hematopoiesis. 25 Recent studies have challenged the dogma that all HSCs express the CD34 antigen. [26][27][28][29][30] Sato and colleagues 31 have demonstrated that only one class of murine stem cell exists, although in 2 functional states distinguishable by CD34 expression. According to this model, CD34 Ϫ stem cells are quiescent and can be activated by different stimuli to generate a CD34 ϩ cell population with high engraftment potential. 32,33 In this study, we assessed the expression of P2Rs on highly purified CD34 ϩ HSCs and investigated the functional responses of Materials and methods ReagentsATP, UTP, cytidine triphosphate (CTP), guano...

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLC␤ 1 , which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLC␤ 1 exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLC␤ 1 subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLC␤ 1 that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLC␤ 1 signaling in G 1 progression by means of a specific target, i.e. cyclin D3/cdk4.It is demonstrated that an autonomous intranuclear inositide cycle exists and that nuclear PLC␤ 1 1 is a key enzyme for cell proliferation and differentiation (1). The enzymes of polyphosphoinositide turnover occur in the nucleus of mammalian cells and yeast as well (Ref. references therein),and there is evidence for phosphatidylinositol bisphosphate (PIP 2 ) synthesis and degradation in the nuclear matrix (3). The evidence obtained with confocal and electron microscope analysis indicates that enzymes required for the synthesis and hydrolysis of phosphoinositides are localized at ribonucleoprotein structures of the inner nuclear matrix involved in transcript processing within the interchromatin domains (4). Although phosphatidylinositol cycle is activated only in nuclei from HeLa cells in S phase (5), striking changes occur mainly in PLC␤ 1 activity a few minutes after growth factor stimulation (1). PLC␤ 1 is composed of two subtypes, 150-kDa PLC␤ 1 a and 140-kDa PLC␤ 1 b, that are derived from a single gene by alternative RNA splicing (6). The two forms of the PLC␤ 1 are detectable both in cytosolic and nuclear fractions although PLC␤ 1 b exists almost entirely in the nucleus (7), and the ␤ 1 a form localizes in equal amount in nuclei and plasma membrane (8). Previous investigations from our group have demonstrated that the nucleus-confined PLC␤ 1 is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells even in the presence of inducers of erythroid differentiation, possibly due to a continuous stimulation of the cell cycle (9). With the above in min...

Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow-derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7% ؎ 2.9% and 42.5% ؎ 3.4%, respectively. Cell recoveries and viabilities were 38.7% ؎ 2.9%, 44.5% ؎ 3.9% and 91.4% ؎ 1.3%, 94.31% ؎ 0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4% ؎ 2.9% (U-23) and 16.6% ؎ 1.4% (C-17) compared with 3.6% ؎ 2.4% and 5.4% ؎ 3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p < .005 for all comparisons). Nucleofection did not affect the immunophenotype of hMSCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents. STEM CELLS 2006;24:454 -461

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The online version of this article has a Supplementary Appendix. BackgroundThe immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. Design and MethodsLeukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. ResultsWe demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4 ConclusionsThese data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.Key words: acute myeloid leukemia, dendritic cells, T regulatory cells, immunotherapy, indoleamine 2,3-dioxygenase.Citation: Curti A, Trabanelli S, Onofri C, Aluigi M, Salvestrini V, Ocadlikova D, Evangelisti C, Rutella S, De Cristofaro R, Ottaviani E, Baccarani M, and Lemoli RM. Indoleamine 2, leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells. Haematologica 2010;95(12):2022-2030. doi:10.3324/haematol.2010 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells

In animals, the bone marrow (BM) is a source of liver-repopulating cells with therapeutic potential in case of tissue damage. However, the early response of human BM-derived stem cells (SC) to liver injury is still unknown. Here, we studied 24 patients undergoing orthotopic liver transplantation (OLT) for end-stage liver disease or hepatocellularcarcinoma, and 13 patients submitted to liver resection. The concentration of circulating BM-derived SC was determined by phenotypic analysis and clonogenic assays. Moreover, we assessed the serum level of inflammatory and tissue-specific cytokines. Reverse transcriptase-polymerase chain reaction and fluorescence-in situ hybridization were also used to characterize mobilized SC. At baseline, patients showed a significant lower concentration of circulating CD133 ؉ , CD34 ؉ SC and clonogenic progenitors (colony-forming unit cells) than healthy controls. However, the time-course evaluation of peripheral blood cells after OLT demonstrated the significant early mobilization of multiple subsets of hematopoietic and endothelial stem/progenitor cells. Cytogenetic and molecular analyses of CD34 ؉ cells showed the host origin of mobilized SC and the expression of transcripts for GATA-4, cytokeratin 19, and ␣-fetoprotein hepatocyte markers. In contrast with OLT, only total circulating CD34 ؉ cells significantly increased after liver resection. Mobilization of BM cells after OLT or liver surgery was associated with increased serum levels of granulocyte-colony stimulating factor, interleukin-6, stem cell factor, hepatocyte growth factor, and vascular endothelial growth factor. In summary, we demonstrate that tissue damage after OLT and liver resection induces increased serum levels of multiple cytokines but only ischemia/reperfusion injury associated with OLT results in the remarkable mobilization of BM stem/progenitor cells. STEM

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the L L family of PI-, and L L4), insulin only activated the L L1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [32 P]orthophosphate, we ascertained that nuclear PI-PLC-L L1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-L L1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-L L1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway. ß