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We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells,using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacologic inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment,and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However,it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2,acting downstream of Kit and other RTKs,promotes Kit gene expression,constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM,spleen,and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors,and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing,self-renewal,and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore,this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals,at least in part through a kinase-phosphatase-kinase cascade. View Publication

U937 monocytic cells are shown here to express a range of PDE4,cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes,PDE4A4,PDE4D5 and PDE4D3,plus the short isoenzyme,PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A,PDE4B and PDE4D activities were 0.63+/-0.09,8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor,rolipram,inhibited immunopurified PDE4B and PDE4D activities similarly,with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast,rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion,which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis,it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes. View Publication

AbstractBackgroundChimeric antigen receptor (CAR) therapies have demonstrated potent efficacy in treating B-cell malignancies,but have yet to meaningfully translate to solid tumors. Nonetheless,they are of particular interest for the treatment of glioblastoma,which is an aggressive form of brain cancer with few effective therapeutic options,due to their ability to cross the highly selective blood-brain barrier.MethodsHere,we use our pooled screening platform,CARPOOL,to expedite the discovery of CARs with antitumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×106 third generation CARs targeting IL-13Rα2,a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity,proliferation,memory formation,and persistence on repeated antigen challenge.ResultsEach enriched CAR robustly produced the phenotype for which it was selected,and one enriched CAR triggered potent cytotoxicity and long-term proliferation on in vitro tumor rechallenge. It also showed significantly improved persistence and comparable tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma,but also demonstrated increased off-target recognition of IL-13Rα1.ConclusionTaken together,this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date. View Publication

OBJECTIVE: The role of semaphorins in tumor progression is still poorly understood. In this study,we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination. METHODS AND RESULTS: We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells,(2) systemic expression of SEMA3A following liver gene transfer in mice,and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings,SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis,without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo. CONCLUSIONS: In sum,both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover,SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule. View Publication

The aorta-gonad-mesonephros region plays an important role in hematopoietic stem cell (HSC) development during mouse embryogenesis. The vascular endothelial cadherin�?� CD45�?� (VE-cad�?�CD45�?�) population contains the major type of immature pre-HSCs capable of developing into long-term repopulating definitive HSCs. In this study,we developed a new coaggregation culture system,which supports maturation of a novel population of CD45-negative (VE-cad�?�CD45�?�CD41�?�) pre-HSCs into definitive HSCs. The appearance of these pre-HSCs precedes development of the VE-cad�?�CD45�?� pre-HSCs (termed here type I and type II pre-HSCs,respectively),thus establishing a hierarchical directionality in the developing HSC lineage. By labeling the luminal surface of the dorsal aorta,we show that both type I and type II pre-HSCs are distributed broadly within the endothelial and subendothelial aortic layers,in contrast to mature definitive HSCs which localize to the aortic endothelial layer. In agreement with expression of CD41 in pre-HSCs,in vivo CD41-Cre-mediated genetic tagging occurs in embryonic pre-HSCs and persists in all lymphomyeloid lineages of the adult animal. View Publication

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

FMS-related tyrosine kinase 3 ligand (FLT3L),encoded by FLT3LG,is a hematopoietic factor essential for the development of natural killer (NK),B cells,and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant,with a history of various recurrent infections,including severe cutaneous warts. The patients’ bone marrow was hypoplastic,with low levels of hematopoietic progenitors,particularly myeloid and B-cell precursors. Counts of B cells,monocytes,and DCs were low in the patients’ blood,whereas the other blood subsets,including NK cells,were affected only moderately,if at all. The patients had normal counts of Langerhans cells and dermal macrophages in the skin but lacked dermal DCs. Thus,FLT3L is required for B-cell and DC development in mice and humans. However,unlike its murine counterpart,human FLT3L is required for the development of monocytes but not NK cells. View Publication

Substantial progress has been made in the development of radiation countermeasures,resulting in the recent approval of several mitigators; however,there has yet to be an approved prophylactic radioprotectant. Research on countermeasure performance in mixed neutron and gamma radiation fields has also been scarce. Fibroblast-stimulating lipopeptide (FSL-1) is a novel synthetic agonist for toll-like receptor 2/6. In previous studies,the administration of FSL-1 before and after gamma radiation significantly improved survival outcomes for mice through the activation of the NF-κB pathway. In the current study,we tested FSL-1’s radioprotective abilities in a mixed radiation field that models one produced by a nuclear detonation in 11–14-week-old C57BL/6 male and female mice. We demonstrate that a single dose of 1.5 mg/kg of FSL-1 administered 12 h prior to 65% neutron 35% gamma mixed-field (MF) irradiation enhances survival,accelerates recovery of hematopoietic cell and stem cell populations,reduces inflammation,and protects innate immune function in mice. FSL-1’s ability to recover blood and protect immune functions is important in countering the high rate of incidence of sepsis caused by MF radiation’s damaging effects. These results demonstrate that FSL-1 is a promising prophylactic countermeasure where exposure to MF radiation is anticipated. View Publication

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity,we developed a fluorescent substrate for ALDH,termed BODIPY aminoacetaldehyde (BAAA),and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells,40-90% pure for CD34(+)CD38(lo/-) cells,and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together,these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well. View Publication

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast,an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor,c-Mpl,regulate primitive hematopoietic populations,including bone marrow hematopoietic stem cells,we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC),TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions,TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies,as well as endothelial cells,confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation,suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation. View Publication