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International audience; Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a Upsilon-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proinflammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242.

Introduction Citrulline, a non-essential amino acid produced exclusively by enterocytes in the small intestine and involved in the synthesis of L-arginine, is not metabolized by the liver. Therefore citrulline serum concentration is highly correlated with functional enterocyte mass, and decreases with digestive toxicity induced by conditioning therapy (radiotherapy and/or chemotherapy) for hematopoietic stem cell transplantation (HSCT) . Acute Graft-versus-host disease (GvHD), one of the major complications of HSCT, is correlated to conditioning-induced gut barrier damage and may be predicted by pre-transplant serum citrulline level (Rashidi, BBMT 2018). It could be interesting to know whether citrulline kinetics could also represent a biomarker for conditioning toxicity, non-relapse mortality (NRM), and GvHD. The aim of this study is thus to define group-based trajectory modeling, to identify clusters of individual serum citrulline kinetics in the early phase of allogeneic HCST, and to test whether these unsupervised trajectories were correlated with these early complications. Materials and Methods Serum citrulline was quantified by liquid chromatography in blood samples collected from consecutive patients who received an allogeneic HSCT in our institution between July 2014 and November 2019. These samples were drawn at different time-points: pre-transplant (D-7, D-3); day of transplant (D0), and post-transplant (D7, D15, D21). Distinct trajectories were identified for serum citrulline by using the semiparametric mixture model described by Nagin (Nagin, Stat Methods Med Res 2018). Results Among 161 patients (pts) included in the study, with a median age of 53 years (17-72), 98 pts (60.9%) received a reduced-intensity conditioning (RIC), 36 pts (22.4%) reduced-toxicity conditioning (RTC), 18 pts (11.1 %) sequential conditioning, and 9 pts (5.6%) myeloablative conditioning (MAC). Donor were identical sibling (22%), matched unrelated donor (52%) and haploidentical sibling (25%). Graft source was peripheral blood mononuclear cells in 144 pts (89.4%) and bone marrow in 17 pts (10.6%) respectively. HCT-CI score was low, intermediate and high-risk in 38%, 32%, and 30% of pts respectively. Disease-Risk Index (DRI) was low/intermediate in 111 pts (69%) and high/very-high in 50 pts (31%). With a median follow up of 29.1 month, 3-year overall survival (OS), disease-free survival (DFS), and NRM rates were 64.5%, 58.3%, and 18.9%, respectively. The median number of citrulline samples per patient was 7 [3-16]. Median citrulline concentrations before conditioning and at D-3, D0, D7 and D15 were statistically different during RIC, RTC, MAC, and sequential conditioning (p In the whole cohort, 3 citrulline trajectories were determined in an unsupervised method. Patients belonging to these 3 trajectories were different according to intensity of conditioning received with lower citrulline trajectories during MAC and sequential conditioning (p After restricting the analysis to pts who received RIC conditioning (n=98), higher pre-HSCT citrulline concentrations were associated with a lower NRM (p=0.042). Unsupervised analysis in this setting individualized 4 clusters of individual trajectories (figure 1), that did neither distinguish age (p=0.28), DRI (p=0.87), HCT-CI score (p=0.81) nor the incidence of acute (p=0.6) or chronic (p=0.4) GvHD. However, the lowest citrulline trajectory contained significantly more haploidentical transplantations (p=0.004) and less pts who received antithymocyte globulin for GvHD prophylaxis (p=0.005). Interestingly in this RIC cohort, cumulative incidence of NRM at 12 months was 23%, 21%, 8%, and 0% respectively according to the 4 citrulline trajectories (figure 2). Conclusion In patients receiving allogeneic HSCT, the variation of serum citrulline concentrations depends on the intensity of the conditioning regimen. In patients who received RIC conditioning, lower plasma citrulline trajectories are associated with higher NRM. In this setting, citrulline may be an attractive biomarker for predicting conditioning toxicity and NRM. Disclosures Hunault: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Diachi: Membership on an entity's Board of Directors or advisory committees; Jansen: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Thepot:astellas: Honoraria; novartis: Honoraria; sanofi: Honoraria; celgene: Honoraria.

Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to >50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts No severe GT-related adverse events have occurred to date. Replication-competent lentivirus was not detected. Analysis of integration site distributions in five subjects showed reconstitution to be highly polyclonal, with no clones expanded to >20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34

Nine Fanconi anemia patients complementation group A (FA-A), age 2-6 years, have been infused with autologous hematopoietic cells after genetic correction with the therapeutic PGK-FANCA.Wpre* lentiviral vector. In all instances patients underwent CD34+ cell mobilization with G-CSF and plerixafor and were subsequently infused in the absence of any pre-conditioning regimen, in order to avoid genotoxic side effects in a population characterized by DNA repair defects and cancer predisposition. The first four patients were treated between January 2016 and March 2017 and were infused with an estimated number of 170,000 and 410,000 transduced CD34+ cells/Kg. The other five patients were treated more recently with cell numbers that ranged between 50,000 to 1.6x106 corrected CD34+ cells/kg. The analyses of the first four patients showed the presence of corrected cells both in BM and PB after six months post-infusion and progressive increases of gene marking were observed thereafter in all these patients until the most recent follow-up (2 to >3 years post-infusion). Gene marking in BM CD34+ cells correlated with the survival of the CFCs to mitomycin-C, with levels up to 70% at 3 years post-infusion. Additionally, progressive decreases in the percentage of PB T cells with diepoxybutane-induced chromosomal breaks were observed in the patients with higher levels of gene marking. Similarly, stabilized PB cell counts have been observed in patients with higher percentages of gene corrected cells. Insertion site analyses revealed the absence of genotoxic events, and demonstrated the engraftment of pluripotent HSCs and a pattern of oligoclonal reconstitution, consistent with the number of infused corrected CD34+ cells and the absence of conditioning. In the five additional patients treated more recently, the presence of gene corrected PB cells has been confirmed; levels of gene marking have been consistent with data observed in the first four treated patients and with the number of infused CD34+ cells. Our results confirm the engraftment of gene corrected HSCs in non-conditioned FA-A patients, in some cases through more than 3 years of follow-up, suggesting the relevance of this therapeutic approach in FA. Disclosures Rio: Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Segovia:Rocket Pharmaceuticals, Inc.: Equity Ownership, Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Wang:GeneWerk: Employment. Casado:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Galy:Genethon: Employment. Cavazzana:SmartImmune: Other: Founder. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH, Heidelberg, Gemrany: Equity Ownership; German Cancer Research Center, Heidelberg, Germany: Employment. Díaz de Heredia:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sevilla:Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

25 mg of iron are required every day to sustain hematopoiesis in the bone marrow. Most iron is consumed by erythroid cells that take up transferrin-bound iron to satisfy their demand for hemoglobin biogenesis. Unexpectedly, erythroblasts express high levels of the iron exporter Ferroportin (Fpn). Fpn surface expression and activity is controlled by Hepcidin, a small liver peptide hormone, produced in response to elevated systemic and tissue iron availability. Resistance of ferroportin to hepcidin binding is caused by a gain of function mutation in the FpnC326S residue, which is the reason for iron overload in patients with Hereditary Hemochromatosis type 4. Some years ago we generated the corresponding mouse model hallmarked by this constitutively active iron exporter. We now applied bone marrow transplantation to investigate the role of the hepcidin/ferroportin regulatory system in cell types of the bone marrow (BM-FpnC326S). CD45.1 host mice were irradiated twice with 500 cGy 4h apart and injected with 2 million of BM cells obtained from C57BL/6N mice (control) or from C57BL/6N congenic constitutive FpnC326S animals. Mice were analyzed three months after transplantation, whereby only animals with an engraftment higher than 95% were included for further analyses. Analysis of the erythroid hematological parameters revealed mild macrocytosis in the presence of unaltered red blood cell count (RBC), hematocrit (HCT), hemoglobin (Hb) and mean corpuscular hemoglobin (MCH) values. Serum iron content, transferrin saturation and serum hepcidin levels remained unchanged, despite a strong decrease in splenic iron levels within the red pulp, where reticuloendothelial macrophages are located. The latter phenotype is consistent with high ferroportin surface expression in macrophages derived from FpnC326S monocytes that causes increased iron export and cellular iron deficiency. Cytokine production (IL1β, TNFα and IL6) in the spleen was unchanged suggesting that iron deficiency in splenic macrophages did not cause inflammation. In contrast to the spleen iron content was not changed in other organs analyzed, including the liver, suggesting that de novo monocyte infiltration is not a major feature in these organs. Interestingly, histological analysis of the femurs revealed a marked decrease in bone marrow iron content; we were unable to detect a single iron-stained cell in the bone marrow of BM-FpnC326S mice, contrasting results from control mice. To investigate whether iron deficiency in bone marrow cells alters numbers of immune cells, we performed a detailed characterization by FACS. Unexpectedly, iron deficiency in the bone marrow did not cause changes in total bone marrow cellularity, total numbers of Ter119+ erythroid cells and the percentage of different erythroblast subpopulations, the number of hematopoietic stem cells (HSC) and of common lymphoid (CLP), myeloid (CMP) and megakaryocyte/erythroid (MEP) progenitors. Taken together, our data clearly demonstrate that at steady state, the hepcidin/ferroportin regulatory circuitry in the bone marrow is dispensable and that iron deficiency in bone marrow cells is not altering the normal hematopoietic process. Future studies will have to extend the analysis to stress conditions. Disclosures Muckenthaler: Novartis: Research Funding; Silence Therapeutics: Consultancy.

Fanconi anemia (FA) is a DNA repair syndrome characterized by bone marrow failure, congenital abnormalities and cancer predisposition. Based on previous experimental results showing the in vivo proliferative advantage of gene corrected FA patients' hematopoietic stem cells (HSCs; Rio, Navarro et al. Blood 2017) a gene therapy trial in non-conditioned FA-A patients was initiated in 2016. Six patients have been treated to-date using fresh and cryopreserved CD34+ cells mobilized to peripheral blood with G-CSF and plerixafor, and transduced with the PGK-FANCA.Wpre* lentiviral vector. Cell doses infused in four patients with a follow-up of at least 12 months varied from 0.6 to 1.4 million CD34+ cells/kg. Transduction efficacies of these samples, determined as vector copies per cell, ranged from 0.17 to 0.53 copies/cell. Despite the absence of patients' conditioning, a marked in vivo expansion of gene-corrected cells was observed in all hematopoietic cell lineages analyzed in BM and PB. Significantly, up to 44% of corrected cells were determined in total PB cells at the most recent follow-up visit (24 month) in the first treated patient. Insertion site analyses in PB cells showed an oligoclonal pattern of hematopoietic reconstitution, and revealed engraftment of multipotent corrected HSCs and no evidence of insertion-site mediated clonal expansion. Functional studies showed significant increases in the resistance of BM progenitors to mitomycin C in all treated patients. Additionally, patients with higher levels of corrected cells also showed significant increases in the chromosomal stability of T cells exposed to diepoxybutane. Finally, analyses discriminating the presence of corrected and uncorrected PB cells in these patients showed marked increases in the total number of corrected leukocytes, contrasting to progressive decreases of uncorrected cells. Our studies demonstrate for the first time that lentiviral-mediated gene therapy results in progressive engraftment and phenotypic correction of HSCs in non-conditioned FA patients, suggesting that this gene therapy approach may constitute a low-toxicity option for the treatment and prevention of BMF in patients with FA. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.

The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway.

The JAKV617F mutation is responsible for the majority of breakpoint cluster region (BCR)/Abelson (ABL)–negative myeloproliferative disorders. Ongoing clinical trials of Janus kinase 2 (JAK2) inhibitors in myeloproliferative disorder patients use small molecules targeting both wild-type and mutated JAK2. To selectively target malignant cells, we developed JAK2V617F-specific small interfering RNAs or short hairpin RNAs. Expression of these RNAs in cell lines or CD34+ cells from patients reduced JAK2V617F-driven autonomous cell proliferation. Mechanisms of inhibition involved selective JAK2V617F protein down-regulation, and consequently, decrease in signal transducer and activator of transcription 5 phosphorylation, cell-cycle progression, and cell survival. However, the addition of high concentrations of cytokines to cell lines or erythropoietin to patient cells greatly reduced growth inhibition. Similarly, the efficacy of a JAK2 small molecule inhibitor on cell line and patient cell proliferation dose dependently decreased with the addition of cytokines. Our results demonstrate that it is possible to specifically target JAK2V617F by RNA interference (RNAi) strategies. In addition, cytokines partially reverse the inhibition induced by both RNAi and small molecule approaches. This strongly suggests that patient cytokine levels in current JAK2 inhibitor clinical trials modulate the outcome of these therapies.

Regulators of G-protein signaling (RGS) constitute a family of proteins involved in the negative regulation of signaling through heterotrimeric G protein–coupled receptors (GPCRs). Several RGS proteins have been implicated in the down-regulation of chemokine signaling in hematopoietic cells. The chemokine stromal-cell–derived factor 1 (SDF-1) activates migration of hematopoietic progenitors cells but fails to activate mature megakaryocytes despite high levels of CXC chemokine receptor 4 (CXCR4) receptor expression in these cells. This prompted us to analyze RGS expression and function during megakaryocyte differentiation. We found that RGS16 and RGS18 mRNA expression was up-regulated during this process. Overexpressing RGS16 mRNA in the megakaryocytic MO7e cell line inhibited SDF-1–induced migration, mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) activation, whereas RGS18 overexpression had no effect on CXCR4 signaling. Knocking down RGS16 mRNA via lentiviral-mediated RNA interference increased CXCR4 signaling in MO7e cells and in primary megakaryocytes. Thus, our data reveal that RGS16 is a negative regulator of CXCR4 signaling in megakaryocytes. We postulate that RGS16 regulation is a mechanism that controls megakaryocyte maturation by regulating signals from the microenvironment.

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1. (Blood. 2005;106: 2580-2589)

Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon γ (IFN-γ) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-γ secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses. (Blood. 2004;104:3267-3275)

The historical model of hematopoiesis, mainly derived from murine studies, is based on the existence of a single hematopoietic stem cell (HSC) capable of generating all blood cell lineages. However this model has been challenged by the proposal of four types of murine HSC, which differ by their relative contribution to the myeloid and lymphoid lineages. Here we have used data from a gene therapy trial to treat Wiskott-Aldrich syndrome (WAS) to explore hematopoiesis in humans. In the trial, the therapeutic vector (lentivirus) integrates into the genome at unique positions in each hematopoietic stem and progenitor cell (HSPC) and is consequently transmitted to all its progeny. Thus hematopoietic ontogeny in humans can be inferred by tracking the appearance of unique integration sites in fractionated blood cell populations. This provides a unique opportunity to model the developmental complexity of the human haematological system. Considerable effort over the last 15 years has been devoted to optimizing retroviral integration sites (RIS) analysis using ligation mediated PCR (LM-PCR), combined with acoustic shearing and high-throughput Illumina sequencing. Acoustic shearing enables more precise quantification of RIS abundance through the enumeration of the various sizes of shear fragments (SonicAbundance) containing a given RIS, which correspond to individual ancestor HSPC and its blood progeny. In four WAS patients treated by gene therapy, we have sorted peripheral blood samples for 5 cell types: myeloid (granulocytes and monocytes) and lymphoid subpopulations (T, B and NK cells), and analysed their RIS profile. Each RIS corresponds to a particular stem/progenitor cell clone, with a particular pattern defined by its presence or absence in each of the 5 lineages. These data are then use to reconstruct aspects of the hematopoietic hierarchy. In order to face the challenging issue of cell sorting contamination we have been using a stringent sort precision mode and we treat residual contamination explicitly in downstream statistical models. Using these approaches, we have characterized up to tens of thousands RIS per patients with a follow up of 4 years. In order to minimize biases due to sparse sampling, we concentrate on the more abundant RIS clones that can be more easily caught and are therefore analysed more reliably. We showed that a significant fraction of RIS clones are detected in a single lineage, while other RIS clones are characterized by different levels of contribution to the myeloid and lymphoid lineages, highlighting the heterogeneity of human HSC. Clones contributing to all 5 lineages are readily recovered but this study also unravels a diversity of inferred hematopoietic programs with various potentials contributing to human blood homeostasis. Longitudinal analysis of clonal dynamics is ongoing, with preliminary results highlighting the maintenance of this heterogeneity of HSPC over time. These new findings provide unique data on human hematopoiesis based on gene corrected WAS patients. Disclosures No relevant conflicts of interest to declare.

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7, a mutant dominant-negative Ikaros protein. In contrast, Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore, Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus, distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137)

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

Pathologies associated with increased circulating iron levels include primary iron overload disorders, such as hereditary hemochromatosis, as well as secondary iron overload disorders, such as thalassemia, sickle cell anemia and myelodysplastic syndromes. In the latter elevated circulating iron levels due to enhanced intestinal iron absorption in support of ineffective erythropoiesis are further increased by the need of chronic blood transfusions. Organ toxicity and increased patient mortality under these conditions are mainly attributed to iron overload and the occurrence of non-transferrin-bound iron (NTBI). So far, epidemiological data and studies in animal models have provided conflicting evidence regarding a role of excess iron in atherogenesis and thrombosis. Here we investigate the role of high circulating iron levels in the development of cardiovascular disease. A mouse model of type IV hereditary hemochromatosis, in which the hepcidin/ferroportin regulatory circuitry is disrupted due to a point mutation in the iron exporter ferroportin (Altamura et al., Cell Metabolism 2014), was interbred with ApoE-null mice, to study its susceptibility to atherosclerosis. As expected, this novel mouse model shows close to 100% transferrin saturation as well as NTBI. The mice were studied at 3, 6 and 12 months of age. We observed a mildly decreased lesion size and number at 3 months of age (6.15±1.25 vs 1.84±0.86 % aortic lesion area, P=0.02), while the same parameters were strongly increased in 6 to 12 month-old mice (6 months: 1.44±0.23 vs 5±0.53 % aortic lesion area, P=0.0001; 12 months: 10.24±1.21 vs 20.44±2.69 % aortic lesion area, P=0.0065). The atherosclerotic phenotype positively correlates with higher levels of circulating iron (12 months: 122.2±6.57 vs 337±19.22 mg iron/dl serum, P Our data suggest that in young mice (3 months) a less severe atherosclerotic phenotype may be explained by iron depletion of macrophages. With increasing age, high circulating iron levels strongly enhance the severity of the atherosclerotic phenotype, indicating that systemic iron overload is a risk factor for atherosclerosis progression and predisposes to cardiovascular disease. Our findings have potential implications for those pathological conditions with elevated systemic iron levels, ranging from patients with hemochromatosis to anemic patients dependent on chronic blood transfusions, as well as for individuals subjected to intravenous iron administration (e.g. patients undergoing hemodialysis). Disclosures No relevant conflicts of interest to declare.

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

It has been proposed that the CD7 molecule is the first antigen expressed on the membrane of cells committed to the T-cell lineage during human fetal T-cell ontogeny. To further identify the pre-T cell subpopulation that migrates to the thymus early in ontogeny, we analyzed the phenotypic and functional characteristics of the fetal liver populations separated on the basis of CD7 expression. Three populations expressing different levels of CD7 were observed: CD7bright, CD7dull, and CD7-. A CD7bright population depleted of mature T, B, and myeloid cells (lineage negative, lin-) and mostly composed of CD56+ CD34- natural killer cells did not mature into T cells in a fetal thymic organ culture (FTOC) assay and was devoid of myeloid progenitors in a clonal colony-forming cell assay. In contrast, the CD7-/dull CD34+ lin- populations were capable of differentiating into phenotypically mature T cells after injection into FTOC and contained early myeloid progenitors. Here we phenotypically compared the fetal liver CD7 populations with the most immature fetal thymic subset that differentiated in the FTOC assay, namely the triple negative (TN, CD3- CD4-CD8-) thymocytes. Fetal TN lin- expressed high levels of CD34 marker and were further subdivided by their expression of CD1 antigen, because CD1- TN thymocytes express higher levels of CD34 antigen compared with CD1+ TN cells. CD1- lin -TN thymocytes are characterized by expressing high levels of CD2, CD7, and CD34 markers and dull levels of CD5, CD10, and CD28 molecules. We could not find fetal liver pre-T cells with a phenotype equivalent to that of TN thymocytes. Our data show that CD7 does not necessarily identify T-cell precursors during fetal T-cell development and strongly support the hypothesis that the acquisition of early T-cell markers as CD2, CD28, and CD5 molecules on the cell surface of T-cell progenitors takes place intrathymically.

Mice with total and constitutive iron regulatory protein 2 (IRP2) deficiency exhibit microcytosis and altered body iron distribution with duodenal and hepatic iron loading and decreased iron levels in splenic macrophages. To explore cell-autonomous and systemic context-dependent functions of IRP2 and to assess the systemic consequences of local IRP2 deficiency, we applied Cre/Lox technology to specifically ablate IRP2 in enterocytes, hepatocytes, or macrophages, respectively. This study reveals that the hepatic and duodenal manifestations of systemic IRP2 deficiency are largely explained by cell-autonomous functions of IRP2. By contrast, IRP2-deficient macrophages from otherwise IRP2-sufficient mice do not display the abnormalities of macrophages from systemically IRP2-deficient animals, suggesting that these result from IRP2 disruption in other cell type(s). Mice with enterocyte-, hepatocyte-, or macrophage-specific IRP2 deficiency display normal red blood cell and plasma iron parameters, supporting the notion that the microcytosis in IRP2-deficient mice likely reflects an intrinsic defect in hematopoiesis. This work defines the respective roles of IRP2 in the determination of critical body iron parameters such as organ iron loading and erythropoiesis.

Mutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the “iron hormone” hepcidin which then controls intestinal iron absorption.