Deborah L. French

Development and Function of Myeloid-Derived Suppressor Cells Generated From Mouse Embryonic and Hematopoietic Stem Cells

Emerging evidence suggests that myeloid‐derived suppressor cells (MDSCs) have great potential as a novel immune intervention modality in the fields of transplantation and autoimmune diseases. Thus far, efforts to develop MDSC‐based therapeutic strategies have been hampered by the lack of a reliable source of MDSCs. Here we show that functional MDSCs can be efficiently generated from mouse embryonic stem (ES) cells and bone marrow hematopoietic stem (HS) cells. In vitro‐derived MDSCs encompass two homogenous subpopulations: CD115+Ly‐6C+ and CD115+Ly‐6C− cells. The CD115+Ly‐6C+ subset is equivalent to the monocytic Gr‐1+CD115+F4/80+ MDSCs found in tumor‐bearing mice. In contrast, the CD115+Ly‐6C− cells, a previously unreported population of MDSCs, resemble the granulocyte/macrophage progenitors developmentally. In vitro, ES‐ and HS‐MDSCs exhibit robust suppression against T‐cell proliferation induced by polyclonal stimuli or alloantigens via multiple mechanisms involving nitric oxide synthase‐mediated NO production and interleukin (IL)‐10. Impressively, they display even stronger suppressive activity and significantly enhance ability to induce CD4+CD25+Foxp3+ regulatory T‐cell development compared with tumor‐derived MDSCs. Furthermore, adoptive transfer of ES‐MDSCs can effectively prevent alloreactive T‐cell‐mediated lethal graft‐versus‐host disease, leading to nearly 82% long‐term survival among treated mice. The successful in vitro generation of MDSCs may represent a critical step toward potential clinical application of MDSCs. STEM CELLS 2010;28:620–632

Distinct STAT Structure Promotes Interaction of STAT2 with the p48 Subunit of the Interferon-α-stimulated Transcription Factor ISGF3

Cells express a variety of STAT (signal transducer and activator of transcription) transcription factors that are structurally homologous and yet function specifically in response to particular cytokines. The functions of the individual STATs are dependent on distinct protein-protein interactions. STAT1 and STAT2 are activated by tyrosine phosphorylation in response to type I interferons-α/β (IFN-α/β) and subsequently form a multimeric transcription factor designated the IFN-α-stimulated gene factor 3 (ISGF3). ISGF3 is a unique STAT complex because it also contains a non-STAT molecule, p48, which is a critical DNA-binding component. We provide evidence that STAT2 specifically interacts with p48 in vivo before and after IFN-α stimulation. The specificity of ISGF3 formation is therefore a result of the distinct nature of the STAT2 molecule. Coimmunoprecipitation assays demonstrate p48 association with STAT2 but not STAT1. Hybrid STAT2·STAT1 molecules were used to identify a region of STAT2 which specifically associates with p48. The region of STAT2 interaction spans an amino-terminal region of two predicted coiled coils. The studies demonstrate thein vivo existence of a STAT2·p48 complex and a distinct STAT2·STAT1 complex after IFN-α stimulation. Data suggest that distinct bipartite complexes STAT2·p48 and STAT2·STAT1 translocate to the nucleus and associate on the DNA target site as ISGF3.

CC Chemokine I-309 Is the Principal Monocyte Chemoattractant Induced by Apolipoprotein(a) in Human Vascular Endothelial Cells

BACKGROUNDnLipoprotein(a) [Lp(a)] is a risk factor for atherosclerosis; however, the mechanisms are unclear. We previously reported that Lp(a) stimulated human vascular endothelial cells to produce monocyte chemotactic activity. The apolipoprotein(a) [apo(a)] portion of Lp(a) was the active moiety.nnnMETHODS AND RESULTSnWe now describe the identification of the chemotactic activity as being due to the CC chemokine I-309. The carboxy-terminal domain of apo(a) containing 6 type-4 kringles (types 5 to 10), kringle V, and the protease domain was demonstrated to contain the I-309-inducing portion. Polyclonal and monoclonal anti-I-309 antibodies as well as an antibody against a portion of the extracellular domain of CCR8, the I-309 receptor, inhibited the increase in monocyte chemotactic activity induced by apo(a). I-309 antisense oligonucleotides also inhibited the induction of endothelial monocyte chemotactic activity by apo(a). I-309 mRNA was identified in human umbilical vein endothelial cells. Apo(a) induced an increase in I-309 protein in the endothelial cytoplasm and in the conditioned medium. Immunohistochemical studies have identified I-309 in endothelium, macrophages, and extracellular areas of human atherosclerotic plaques and have found that I-309 colocalized with apo(a).nnnCONCLUSIONSnThese data establish that I-309 is responsible for the monocyte chemotactic activity induced in human umbilical vein endothelial cells by Lp(a). The identification of the endothelial cell as a source for I-309 suggests that this chemokine may participate in vessel wall biology. Our data also suggest that I-309 may play a role in mediating the effects of Lp(a) in atherosclerosis.

Platelet Glycoprotein IIb/IIIa Receptors and Glanzmann’s Thrombasthenia

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 nnPlatelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

The integrin αIIbβ3 and αVβ3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both αIIbβ3 and αVβ3 receptors (4, 5). Abciximab is a mouse/human chimeric Fab fragment of the mAb 7E3 that inhibits αIIbβ3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6). n nPrevious studies of 7E3 binding to cells expressing recombinant αIIbβ3 receptors demonstrated that: (i) swapping select murine for human αIIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156–G189 sequence) from β3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177–C184 sequence within the SDL region in β3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129–T133 sequence for the human W129–N133 sequence results in partial loss of 7E3 binding (7). n nThe above human/mouse swapping studies identified regions within β3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129–N133 region contains two amino acid differences between human β3(β3Hu) and mouse β3 (β3M) and the C177–C184 region contains three amino acid differences (Table 1). To define further the regions on αIIbβ3 that control 7E3 binding, we assessed the effects of individual amino acid substitutions on 7E3 binding to cells expressing αIIbβ3, as well as the effects of these substitutions on receptor biochemistry and function. In addition, we studied the effect of mutating a highly conserved lysine residue (K125) that appears to link the C177–C184 loop to the α-helix containing W129–N133. n n n nTable 1. n nConstructs for mammalian cell expression n n n nFinally, in view of our localization of the region involved in 7E3 binding to the head region of β3 adjacent to the arginine-glycine-aspartic acid (RGD) binding site, our previous studies demonstrating that 7E3 IgG (but not 7E3 Fab) binds much more rapidly to activated than unactivated platelets, (3, 9), and our recently proposed model of αIIbβ3 undergoing a change from a bent to an extended conformation upon activation, (10, 11), we also assessed the binding of 7E3 IgG and 7E3 Fab to an αIIbβ3 receptor reversibly locked in a bent conformation (10).

Stathmin expression and megakaryocyte differentiation:A potential role in polyploidy

OBJECTIVEnMegakaryopoiesis is characterized by two major processes, acquisition of lineage-specific markers and polyploidization. Polyploidy is a result of endomitosis, a process that is characterized by continued DNA replication in the presence of abortive mitosis. Stathmin is a major microtubule-regulatory protein that plays an important role in the regulation of the mitotic spindle. Our previous studies had shown that inhibition of stathmin expression in human leukemia cells results in the assembly of atypical mitotic spindles and abnormal exit from mitosis. We hypothesized that the absence of stathmin expression in megakaryocytes might be important for their abortive mitosis.nnnMATERIALS AND METHODSnThe experimental models that we used were human K562 and HEL cell lines that can be induced to undergo megakaryocytic differentiation and primary murine megakaryocytes generated by in vitro culture of bone marrow cells. The megakaryocytic phenotype was evaluated by flow cytometry and light microscopy. The DNA content (ploidy) was analyzed by flow cytometry. Stathmin expression was analyzed by Western and Northern blotting and by RT-PCR.nnnRESULTSnOur studies showed an inverse correlation between the level of ploidy and the level of stathmin expression in megakaryocytic cell lines and in primary cells. More importantly, inhibition of stathmin expression in K562 cells enhanced the propensity of these cells to undergo endomitosis and to become polyploid upon induction of megakaryocytic differentiation. In contrast, inhibition of stathmin expression interfered with the ability of the cells to acquire megakaryocyte-specific markers of differentiation.nnnCONCLUSIONnBased on these observations, we propose a model of megakaryopoiesis in which stathmin expression is necessary for the proliferation and differentiation of early megakaryoblasts and its suppression in the later stages of megakaryocytic maturation is necessary for polyploidization.

Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation

The integrin αIIbβ3 and αVβ3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both αIIbβ3 and αVβ3 receptors (4, 5). Abciximab is a mouse/human chimeric Fab fragment of the mAb 7E3 that inhibits αIIbβ3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6). n nPrevious studies of 7E3 binding to cells expressing recombinant αIIbβ3 receptors demonstrated that: (i) swapping select murine for human αIIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156–G189 sequence) from β3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177–C184 sequence within the SDL region in β3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129–T133 sequence for the human W129–N133 sequence results in partial loss of 7E3 binding (7). n nThe above human/mouse swapping studies identified regions within β3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129–N133 region contains two amino acid differences between human β3(β3Hu) and mouse β3 (β3M) and the C177–C184 region contains three amino acid differences (Table 1). To define further the regions on αIIbβ3 that control 7E3 binding, we assessed the effects of individual amino acid substitutions on 7E3 binding to cells expressing αIIbβ3, as well as the effects of these substitutions on receptor biochemistry and function. In addition, we studied the effect of mutating a highly conserved lysine residue (K125) that appears to link the C177–C184 loop to the α-helix containing W129–N133. n n n nTable 1. n nConstructs for mammalian cell expression n n n nFinally, in view of our localization of the region involved in 7E3 binding to the head region of β3 adjacent to the arginine-glycine-aspartic acid (RGD) binding site, our previous studies demonstrating that 7E3 IgG (but not 7E3 Fab) binds much more rapidly to activated than unactivated platelets, (3, 9), and our recently proposed model of αIIbβ3 undergoing a change from a bent to an extended conformation upon activation, (10, 11), we also assessed the binding of 7E3 IgG and 7E3 Fab to an αIIbβ3 receptor reversibly locked in a bent conformation (10).

Prenatal diagnosis of Glanzmann thrombasthenia using the polymorphic markers BRCA1 and THRA1 on chromosome 17

Glanzmann thrombasthenia is an autosomal recessive bleeding disorder caused by mutations in the genes encoding platelet GPIIb or GPIIIa. Both genes map to chromosome 17q21 and polymorphisms within this chromosomal region have been identified. In the current study, prenatal diagnosis was performed for a family that already had one affected child, patient 1, who had a compound heterozygous mutation in GPIIb. At the time of prenatal diagnosis, the maternal GPIIb mutation had been identified but the paternal GPIIb mutation was unknown. By sequence analysis, the fetus was identified as a carrier of the mothers mutation. To determine the probability of the fetus inheriting the fathers mutation, haplotype analysis of DNA samples from the fetus, mother, father and affected child were performed using polymorphic markers on chromosome 17q12‐q21. These markers included polymorphisms within the thyroid hormone receptor α1 gene (THRA1), the breast cancer gene (BRCA1), GPIIb, GPIIIa, and an anonymous marker D17S579. Heterozygosity within the THRA1, BRCA1 and GPIIIa polymorphic markers predicted that the fetus carried the fathers normal allele. Based on genetic linkage studies, no recombination was identified with any of the informative markers, and from the map distance between GPIIb and BRCA1 the accuracy of diagnosis was predicted to be >98%. The fathers mutation was subsequently identified and direct sequence analysis of fetal DNA confirmed that the fetus did not inherit the fathers mutant allele.