Loss of the fibroblastic reticular cell (FRC) network in lymphoid tissues (LTs) during HIV-1 infection has been shown to impair the survival of naïve T cells and limit immune reconstitution after anti-retroviral therapy (ART). What causes this FRC loss is unknown. Because FRC loss correlates with loss of both naïve CD4 and CD8 T cell subsets and decreased lymphotoxin-beta, a key factor for maintenance of FRC network, we hypothesized that loss of naïve T cells is responsible for loss of the FRC network. To test this hypothesis, we assessed the consequences of antibody-mediated depletion of CD4 and CD8 T cells in rhesus macaques and sooty mangabeys. We found that only CD4 T cell depletion resulted in FRC loss in both species and that this loss was caused by decreased lymphotoxin-beta mainly produced by the CD4 T cells. We further found the same dependence of the FRC network on CD4 T cells in HIV-1 infected patients before and after ART, and in other immunodeficiency conditions such as CD4 depletion in cancer patients induced by chemotherapy and irradiation. CD4 T cells thus play a central role in the maintenance of LT structure necessary for their own homeostasis and reconstitution.

Natural killer (NK) cells play a critical role in early host defense to infected and transformed cells. Here we show that mice deficient in Eri1, a conserved 3'-to-5' exoribonuclease that represses RNA interference, have a cell-intrinsic defect in NK cell development and maturation. Eri1(-/-) NK cells displayed delayed acquisition of Ly49 receptors in the bone marrow and a selective reduction in Ly49D and Ly49H activating receptors in the periphery. Eri1 was required for immune-mediated control of mouse cytomegalovirus (MCMV) infection. Ly49H(+) NK cells deficient in Eri1 failed to expand efficiently during MCMV infection, and virus-specific responses were also diminished among Eri1(-/-) T cells. We identified miRNAs as the major endogenous small RNA target of Eri1 in mouse lymphocytes. Both NK and T cells deficient in Eri1 displayed a global, sequence-independent increase in miRNA abundance. Ectopic Eri1 expression rescued defective miRNA expression in mature Eri1(-/-) T cells. Thus, mouse Eri1 regulates miRNA homeostasis in lymphocytes and is required for normal NK cell development and anti-viral immunity.

One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional SLAM-associated protein (SAP) in T, invariant NKT (NKT) and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whilst NKT cell-dependent B cell responses were absent in SAP(-/-).B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP(-/-).BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted post-development in SAP(fl/fl).tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensible for non-cognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell-mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

The impact of HIV-1 Nef-mediated Human Leukocyte Antigen class I (HLA-I) downregulation on CD8(+) cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. We examine the impact of Nef on the antiviral efficiency of HIV-1 specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef did not correlate with the presenting HLA-I type or functional avidity of CTLs, but related directly to the kinetics of infected cell clearance. While Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that while Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I downregulation, this varies depending on the specific epitope and virus inoculum. Generally reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.

Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that upregulation of Mpl expression in mice induces AML when co-expressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that upregulation of wild type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and anti-angiogenic factors throughout the vascular system. Platelets are anucleated cells and lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments. However, studies have in the past demonstrated the presence of glycosyltransferase activities associated with platelets. We show that members of three distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicate that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Blood platelets are therefore a rich source of both glycosyltransferases and donor sugar substrates, which can be released to function in the extracellular space. This platelet glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biological functions.

The progress made in the understanding of the biology and management of chronic myeloid leukemia (CML) since the recognition of a common chromosomal abnormality to the introduction of ever more effective tyrosine kinase inhibitors (TKI) is unprecedented in cancer. The expected survival for patients diagnosed with CML today, if properly managed and achieving an optimal response, is probably similar to that of the general population, marking a dramatic change in the natural history of the disease. There are better treatment options and better monitoring tools that help us adequately manage patients with CML. In treating these patients it is important to keep in mind the principle that the goal is to achieve the best long-term outcome for them. It is also important to base the treatment decisions based on the data available. The results from cytogenetic and molecular analyses have to be interpreted judiciously and all available treatment options integrated into the treatment plan properly. The availability of several treatment options in CML is an asset, but the temptation of rapid succession of treatment changes because of perceived "suboptimal" response or for adverse events that could be managed needs to be avoided. Any decision to change therapy needs to weight the expected long-term outcome with the current option versus the true expectations with any new option, particularly as it relates to irreversible outcomes such as transformation to blast phase and death. In this manuscript we discuss the treatment approach that has helped us manage successfully a large CML population.

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via two pair of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom, is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with five amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FVMTTS/Y, had increased membrane interaction and apparent membrane-independent activity that was due to phospholipid retained during purification. Phospholipid-free FVMTTS/Y showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in Chronic Lymphocytic Leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biological alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2 and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium/calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced upon IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive. At the opposite, ionomycin-induced NFAT activity allows survival of non-responding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR/NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR/NFAT intermediates as promising functional therapeutic targets.

Fanconi anemia (FA) is a rare bone-marrow failure disorder with defective DNA interstrand cross link repair. Still there are FA patients without mutations in any of the fifteen genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2, and is required for interstrand cross link repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular and genetic features in four patients carrying a homozygous 15q13.3 microdeletion including FAN1 and six additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3'- and 5'-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all four FAN1-deficient patients while their cells showed mild sensitivity to MMC in terms of cell survival and G2 phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand cross link repair when compared to true FA genes and exclude FAN1 as a novel FA gene.

Brentuximab vedotin induces an overall response rate of 75% in patients with relapsed/refractory Hodgkin lymphoma (HL), but its impact on future allogeneic transplantation (allo-HCT) is not known. We retrospectively examined records of 18 patients with relapsed/refractory HL who were treated on brentuximab vedotin clinical trials to evaluate the efficacy and safety of subsequent reduced-intensity allo-HCT. Seventeen patients had previous autologous transplant; 6 were in complete remission, and 8 were in partial remission prior to allo-HCT with 12 grafts from unrelated or mismatched donors. The 1-year overall survival was 100%, progression-free survival was 92.3 % and non-relapse mortality was 0%(median follow up of 14 months). The incidence of acute graft-versus-host disease (GVHD) was 27.8% and chronic GVHD was 56.3%. Brentuximab vedotin prior to reduced-intensity allo-HCT does not appear to adversely affect engraftment, GVHD, or survival, and may provide sufficient disease control to enable reduced-intensity allo-HCT.

The calcineurin/NFAT signaling pathway mediates multiple adaptive T cell functions, but recent studies have shown that calcineurin/NFAT signaling also contributes to innate immunity and regulates the homeostasis of innate cells. Myeloid cells, including granulocytes and dendritic cells, can promote inflammation, regulate adaptive immunity, and are essential mediators of early responses to pathogens. Microbial ligation of pattern-recognition receptors such as TLR4, CD14 and dectin 1 is now known to induce the activation of calcineurin/NFAT signaling in myeloid cells, a finding which has provided new insights into the molecular pathways that regulate host protection. Inhibitors of calcineurin/NFAT binding, such as cyclosporine A and FK506 are broadly used in organ transplantation and can act as potent immunosuppressive drugs in a variety of different disorders. There is increasing evidence that these agents influence innate responses as well as inhibiting adaptive T cell functions. This review focuses on the role of calcineurin/NFAT signaling in myeloid cells, which may contribute to the various unexplained effects of immunosuppressive drugs already being used in the clinic.

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR monoclonal antibody, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs and increased the frequency of Bl-CFCs by up to 10 fold. Furthermore, APLN peptide also synergised with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC derived hematopoietic and endothelial cells.

Anemia is very common in patients suffering from infections or chronic inflammation and can add substantially to the morbidity of the underlying disease. It is mediated by excessive production of the iron-regulatory peptide, hepcidin, but the signaling pathway responsible for hepcidin upregulation in the inflammatory context is still imperfectly known. We show here that activin B has an unexpected but crucial role in the induction of hepcidin by inflammation. There is indeed a dramatic induction of Inhbb mRNA, encoding activin β(B)-subunit, in the liver of mice challenged with lipopolysaccharide, slightly preceding an increase in Smad1/5/8 phosphorylation and Hamp mRNA. Activin B also induces Smad1/5/8 phosphorylation in human hepatoma-derived cells and, synergistically with IL-6 and STAT-3 signaling, markedly upregulates hepcidin expression, an observation confirmed in mouse primary hepatocytes. Pretreatment with a BMP type I receptor inhibitor shows that the effect of activin B on hepcidin expression is entirely attributable to its effect on BMP signaling, most likely via ALK3. Activin B is thus a novel and specific target for the treatment of anemia of inflammation.

Anemia linked to a relative deficiency of renal erythropoietin production is a significant cause of morbidity and medical expenditures in the developed world. Recombinant erythropoietin is expensive and has been linked to excess cardiovascular events. Moreover, some patients become refractory to erythropoietin due to increased production of factors such as hepcidin. During fetal life the liver, rather than the kidney, is the major source of erythropoietin. Here we show that it is feasible to reactivate hepatic erythropoietin production and suppress hepcidin levels using systemically delivered siRNAs targeting the EglN prolyl hydroxylases specifically in the liver, leading to improved red blood cell production in models of anemia caused by either renal insufficiency or chronic inflammation with enhanced hepcidin production.

Polycythemia vera (PV) is a clonal disorder characterized by unwarranted production of red blood cells. In the majority of cases PV is driven by oncogenic mutations that constitutively activate the JAK-STAT signal transduction pathway such as JAK2 V617F or exon 12 mutations or LNK mutations. Diagnosis of PV is based on the WHO criteria. Diagnosis of post-PV myelofibrosis is established according to the IWGMRT criteria. Different clinical presentations of PV are discussed. Prognostication of PV is tailored to the most frequent complication during follow-up, namely thrombosis. Age over 60 years and prior history of thrombosis are the two main risk factors for disease stratification. Correlations are emerging between leukocytosis, JAK2(V617F) mutation, bone marrow fibrosis and different outcomes of PV, which need to be confirmed in prospective studies. In my practice, hydroxyurea is still the gold standard when cytoreduction is needed, even though pegylated interferon-alfa-2a and ruxolitinib might be useful in particular settings. Results of phase 1-2 studies concerning these latter agents should however be confirmed by the ongoing randomized phase 3 clinical trials. In this paper I will discuss the main problems encountered in daily clinical practice with PV patients regarding diagnosis, prognostication and therapy.

Thrombotic microangiopathies (TMAs) are a group of life-threatening disorders characterized by thrombocytopenia, fragmentation of erythrocytes and ischemic organ damage. Genetic disorders, autoimmune disease and cancer are risk factors for TMAs, but an additional, unknown trigger is needed to bring about acute disease. Recent studies suggest that DNA and histones are released during inflammation or infection and stimulate coagulation, thrombosis, thrombocytopenia and organ damage in mice. Here we show that extracellular DNA and histones as well as markers of neutrophils are present in acute TMAs. Analysis of plasma from TMA patients of different clinical categories revealed elevated levels of DNA/histone complexes and myeloperoxidase (MPO) from neutrophil granules as well as S100A8/A9, a heterocomplex abundant in neutrophil cytosol. During therapy of patients with thrombotic thrombocytopenic purpura (TTP), a subtype of TMAs often associated with severe ADAMTS13 deficiency, plasma DNA and MPO were inversely correlated with platelet counts and their levels indicated amelioration or exacerbation of the disease. ADAMTS13 deficiency together with increased levels of plasma DNA and MPO were characteristic for acute TTP. A minor infection often precedes acute TMA and extracellular DNA and histones released during the inflammatory response could provide the second hit, which precipitates acute TMA in patients with pre-existing risk factors.

Platelet dense granules are members of a family of tissue-specific lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules upon platelet activation promote coagulation and hemostasis, and dense granule defects as in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known regarding how dense granules form in megakaryocytes (MKs). Here we exploit SLC35D3, mutations in which cause a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that Slc35d3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from two of three HPS models, and when expressed ectopically in melanocytes SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. Thus SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

The physiological role played by plasmacytoid dendritic cells (pDCs) in the induction of innate responses and inflammation in response to pathogen signaling is not well understood. Here, we describe a new mouse model lacking pDCs and establish that pDCs are essential for the in vivo induction of natural killer (NK) cell activity in response to toll like receptor 9 (TLR9) triggering. Furthermore, we provide the first evidence that pDCs are critical for the systemic production of a wide variety of chemokines in response to TLR9 activation. Consequently, we observed a profound alteration in monocytes, macrophages, neutrophils, and NK cells recruitment at the site of inflammation in the absence of pDCs in response to CpG-Dotap and stimulation by microbial pathogens such as Leishmania major, Escherichia coli, and Mycobacterium bovis. This study, which is based on the development of a constitutively pDC-deficient mouse model, highlights the pivotal role played by pDCs in the induction of innate immune responses and inflammation following TLR9 triggering.