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Natural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL.

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.

Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However, the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study, we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow, in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells, NK progenitors, and NKG2A-negative NK cells), and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore, we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally, we show that acetylated histones are associated with the CpG-rich region in NKG2A positive, but not negative, cell lines, and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription, but cotreatment with both drugs resulted in a significantly greater induction, suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells.

Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However, their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore, we studied the role of Pneumocystis carinii, as well as Saccharomyces cerevisiae, cell wall-derived beta-glucans, in DC costimulatory molecule expression. We further studied the impact of beta-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL), its role in beta-glucan activation of DCs was also investigated. beta-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by beta-glucans elicits T cell activation and polarization into a Th1 patterned response, but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization, suggesting that beta-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1beta and TNF-alpha secretion by beta-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions, further promoting lung inflammation typical of Pneumocystis pneumonia.

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC50 of less than 2 microM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation.

Central issues in intracoronary infusion (ICI) of bone marrow (BM)-cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM-progenitors. We could immunomagnetically isolate 0.8 +/- 0.32 x 10(7) CD133(+) cells and 0.75 +/- 0.24 x 10(7) CD133(-)CD34(+) cells from 310 +/- 40 ml BM. After labeling these cells with (99m)Tc-hexamethylpropylenamineoxime, they were infused into the infarct-related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% +/- 3.6 and 6.8% +/- 2.4 of the total infused radioactivity in the infarcted area of the heart, respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133(+) and CD133(-)CD34(+) BM-progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium.

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients, respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (textgreater or = 98%) for CD73, CD105, CD106, CD29, CD13, CD44 and negative (textless or = 1%) for CD34, CD45, CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients, respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units.