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Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

Neurodevelopmental disorders (NDDs) often result from highly penetrant variation in one of many genes, including genes not yet characterized. Using the MatchMaker Exchange, we assembled a cohort of 22 individuals with rare, protein-altering variation in the X-linked transcriptional coregulator gene ZMYM3. Most (n=19) individuals were males; 15 males had maternally-inherited alleles, three of the variants in males arose de novo, and one had unknown inheritance. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n=21) are missense, two of which are recurrent. Three unrelated males were identified with inherited variation at R441, a site at which variation has been previously reported in NDD-affected males, and two individuals have de novo variation at R1294. All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is highly expressed in the brain, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one mutant, ZMYM3R1274W, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to support a conclusive causative role for variation in ZMYM3 in disease, the totality of the evidence, including the presence of recurrent variation, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally-confirmed functional effects, strongly supports ZMYM3 as a novel NDD gene.

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) has been used to identify transcription factor (TF) binding proteins throughout the genome. Unfortunately, this approach traditionally requires commercially available, ChIP-seq grade antibodies that frequently fail to generate acceptable datasets. To obtain data for the many TFs for which there is no appropriate antibody, we recently developed a new method for performing ChIP-seq by epitope tagging endogenous TFs using CRISPR/Cas9 genome editing technology (CETCh-seq). Here, we describe our general protocol of CETCh-seq for both adherent and nonadherent cell lines using a commercially available FLAG antibody.

Hematopoietic stem cells (HSCs) reside in association with bone marrow (BM) sinusoidal vessels in vivo, but the function of BM endothelial cells (ECs) in regulating hematopoiesis is unclear. We hypothesized that hematopoietic regeneration following injury is regulated by BM ECs. BALB/c mice were treated with total body irradiation (TBI) and then infused with C57Bl6-derived endothelial progenitor cells (EPCs) to augment endogenous BM EC activity. TBI caused pronounced disruption of the BM vasculature, BM hypocellularity, ablation of HSCs, and pancytopenia in control mice, whereas irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, and platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti–VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non–EPC-treated mice compared with irradiated controls. These data demonstrate that allogeneic EPC infusions can augment hematopoiesis and suggest a relationship between BM microvascular recovery and hematopoietic reconstitution in vivo.

Flk-1+ endothelial progenitors contribute critically to the definitive onset of hematopoiesis during embryogenesis. Recent studies have suggested that adult sources of endothelial cells also possess hematopoietic activity. In this study, we sought to determine whether transplantation of primary vascular endothelial cells (ECs) could enhance the hematopoietic recovery and survival of irradiated mice. C57Bl6 mice were exposed to sublethal and lethal doses of irradiation and were subsequently given transplants of either primary murine brain–derived ECs (MBECs) or fetal blood-derived ECs (FBECs). Mice that received a transplant with MBECs alone demonstrated accelerated BM cellular recovery, radioprotection of BM c-kit+sca-1−lin− progenitors and enhanced regeneration of c-kit+sca-1+lin− (KSL) stem/progenitor cells following irradiation compared with controls. MBEC transplantation also facilitated the recovery of circulating white blood cell and platelet counts following radiation exposure. Remarkably, 57% of mice that received a transplant with MBECs alone survived long term following 1050 cGy exposure, which was 100% lethal in control mice. FBEC transplantation was also associated with increased survival compared with controls, although these mice did not survive in the long term. These data suggest that reestablishment of endothelial cell activity can improve the hematopoietic recovery and survival of irradiated mice.

Problem Toll-like receptors (TLRs) recognition is an important means for the innate immune system to rapidly respond to pathogen invasion. Our aim was to determine whether uterine natural killer (uNK) cell cytokine production induced by stimulation through TLRs could be regulated by endogenous transforming growth factor (TGF)-beta in human endometrium. Method of study Single cells were isolated from human endometrium, and interferon (IFN)-gamma production by endometrium cells and uNK cells was determined after stimulation by TLR agonists. The role of TGF-beta in regulating this response was tested by blocking TGF-beta function using antibodies or a specific inhibitor, SB431542. Results TGF-beta blockade increased TLR agonist induced IFN-gamma by uNK cells. The regulation of uNK cell cytokine production was observed when uNK cells were incubated with agonists for TLR2 (PGN) or TLR3 (polyI:C). Blockade of TGF-beta or TGF-beta receptor signaling had no effect on constitutive cytokine production in the absence of TLR agonists. Conclusion The results indicate that endogenous TGF-beta alters cytokine responses of uNK cells in human endometrium in response to TLR agonists. These data suggest that uNK cell responses to microbial pathogens in the endometrium are regulated by the amount of biologically active TGF-beta present within the human endometrium.

Numerous studies have shown that NK cells are important in controlling the early stages of infection with alpha- or betaherpesviruses. In contrast, little is known about the impact of NK cells on gammaherpesvirus infections. We tested mice with defects in NK cells for their ability to resist murine gammaherpesvirus (MHV-68) infection. The depletion of NK cells had no effect on the control of the acute or latent stages of the infection. In addition, transgenic mice deficient in NK cells controlled the infection in a comparable manner to wild-type mice. We also showed that the antiviral CD8 T cell response was unaffected by the presence or absence NK cells. We conclude that NK cells contribute little to the control of MHV-68 infection, and therefore, NK cells are not essential for controlling all herpesvirus infections.

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10−9 M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56bright blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.

Natural killer (NK) cells are a major population of lymphocytes in the human endometrium (EM), and NK cells can be a significant source of cytokines that alter local immune responses. The aim of this study was to determine the expression of NK cell receptors in situ and to test whether uterine NK (uNK) cells produce cytokines and how this activity may be regulated by transforming growth factor-β (TGF-β). We observed that human uNK cells were CD56+, CD3−, CD57−, CD9+, CD94+, killer inhibitory receptor+, and CD16+/− in situ by confocal microscopy. We examined cytokine production by uNK cells and uNK cell clones derived from human EM. Stimulation of uNK cells with interleukin (IL)-12 and IL-15, both of which are expressed in the human EM, induced interferon-γ (IFN-γ) and IL-10 production. IFN-γ production by uNK cell clones was completely inhibited by TGF-β1 in a dose-dependent manner with an inhibitory concentration 50% value of 20 pg/ml. IL-10 secretion by uNK cell clones was also inhibited by TGF-β1 at similar concentrations. Furthermore, blocking endogenous TGF-β in fresh human endometrial cell cultures increased the production of IFN-γ by uNK cells. These data indicate that uNK cells have a unique phenotype that is distinct from blood NK cells. Further, data demonstrate that uNK cells can produce immunoregulatory cytokines and that inhibition of uNK cells by locally produced TGF-β1 is a likely mechanism to regulate NK cell function in the human EM.

Hematopoietic stem cells (HSCs) reside in proximity to bone marrow endothelial cells (BM ECs) and maintenance of the HSC pool is dependent upon EC-mediated c-kit signaling. Here, we used genetic models to determine whether radioprotection of BM ECs could facilitate hematopoietic regeneration following radiation-induced myelosuppression. We developed mice bearing deletion of the proapoptotic proteins, BAK and BAX, in Tie2+ ECs and HSCs (Tie2Bak/BaxFl/− mice) and compared their hematopoietic recovery following total body irradiation (TBI) with mice which retained Bax in Tie2+ cells. Mice bearing deletion of Bak and Bax in Tie2+ cells demonstrated protection of BM HSCs, preserved BM vasculature, and 100% survival following lethal dose TBI. In contrast, mice that retained Bax expression in Tie2+ cells demonstrated depletion of BM HSCs, disrupted BM vasculature, and 10% survival post-TBI. In a complementary study, VEcadherinBak/BaxFl/− mice, which lack Bak and Bax in VEcadherin+ ECs, also demonstrated increased recovery of BM stem/progenitor cells following TBI compared to mice which retained Bax in VEcadherin+ ECs. Importantly, chimeric mice that lacked Bak and Bax in HSCs but retained Bak and Bax in BM ECs displayed significantly decreased HSC content and survival following TBI compared to mice lacking Bak and Bax in both HSCs and BM ECs. These data suggest that the hematopoietic response to ionizing radiation is dependent upon HSC-autonomous responses but is regulated by BM EC-mediated mechanisms. Therefore, BM ECs may be therapeutically targeted as a means to augment hematopoietic reconstitution following myelosuppression.

In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79–100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16–43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34−c-kit+Sca-1+lineage− (34−KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34−KSL cells. Treatment of bone marrow (BM) 34−KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.

Background Previous work has demonstrated the potential for peripheral blood (PB) gene expression profiling for the detection of disease or environmental exposures. Methods and Findings We have sought to determine the impact of several variables on the PB gene expression profile of an environmental exposure, ionizing radiation, and to determine the specificity of the PB signature of radiation versus other genotoxic stresses. Neither genotype differences nor the time of PB sampling caused any lessening of the accuracy of PB signatures to predict radiation exposure, but sex difference did influence the accuracy of the prediction of radiation exposure at the lowest level (50 cGy). A PB signature of sepsis was also generated and both the PB signature of radiation and the PB signature of sepsis were found to be 100% specific at distinguishing irradiated from septic animals. We also identified human PB signatures of radiation exposure and chemotherapy treatment which distinguished irradiated patients and chemotherapy-treated individuals within a heterogeneous population with accuracies of 90% and 81%, respectively. Conclusions We conclude that PB gene expression profiles can be identified in mice and humans that are accurate in predicting medical conditions, are specific to each condition and remain highly accurate over time.

In this study, we have analyzed the presence and subsets of NK cells throughout the tissues of the FRT. We demonstrate that there are NK cells in the various FRT tissues and that their phenotype and regulation are largely dependent upon the FRT tissue where they reside. NK cells in the Fallopian tube, endometrium, cervix, and ectocervix expressed CD9 while blood NK cells did not. We have also found that unique subsets of NK cells are in specific locations of the FRT. The NK cells in the lower reproductive tract did not express CD94, but they did express CD16. In contrast, NK cells in the upper FRT express high amounts of CD94 and CD69, but few NK cells expressed CD16. All of these FRT NK cells were able to produce IFN-gamma upon stimulation with cytokines. Furthermore, the number of NK cells varied with the menstrual cycle in the endometrium but not in the cervix or ectocervix. These data suggest that unique characteristics of the tissues may account of specific localization of different NK cell subsets.

The human endometrium (EM) contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay between these different cell types and their roles in defense against pathogen invasion in this specialized tissue are important for controlling infection and reproduction. TLRs are a family of receptors able to recognize conserved pathogen-associated molecular patterns. In this study, we determined the expression of TLRs on uterine NK (uNK) cells from the human EM and the extent to which uNK cells responded to TLR agonist stimulation. uNK cells expressed TLRs 2, 3, and 4, and produced IFN-γ when total human endometrial cells were stimulated with agonists to TLR2 or TLR3 (peptidoglycan or poly(I:C), respectively). Activated uNK cell clones produced IFN-γ upon stimulation with peptidoglycan or poly(I:C). However, purified uNK cells did not respond directly to TLR agonists, but IFN-γ was produced by uNK cells in response to TLR stimulation when cocultured with APCs. These data indicate that uNK cells express TLRs and that they can respond to TLR agonists within EM by producing IFN-γ. These data also indicate that the uNK cells do not respond directly to TLR stimulation, but rather their production of IFN-γ is dependent upon interactions with other cells within EM.

NK cells are an important component of innate immunity, and they can promote CTL and Th1 cell development and macrophage activation via cytokines. TGF-beta is believed to be an important immunoregulatory molecule, and for this reason several TGF-beta inhibitors are currently in clinical development. However, the modulation of specific innate immune responses by endogenous human TGF-beta remains unclear. In this study, we demonstrate that blocking the action of endogenous TGF-beta resulted in an increase in both the percentage of responding NK cells and the amount of IFN-gamma produced by human NK cells when stimulated by monokines and TLR agonists. Blocking endogenous TGF-beta resulted in significant NK cell IFN-gamma production under suboptimal stimulation conditions. Our findings also suggest that TGF-beta associated with other blood cells may be involved in limiting NK cell activation. Thus, inhibiting endogenous TGF-beta provides a means to shift NK cell activation and promote cellular immunity.

Background: Current evidence indicates that even low-level lead (Pb) exposure can have detrimental effects, especially in children. We tested the hypothesis that Pb exposure alters gene expression patterns in peripheral blood cells and that these changes reflect dose-specific alterations in the activity of particular pathways. Methodology/Principal Finding: Using Affymetrix Mouse Genome 430 2.0 arrays, we examined gene expression changes in the peripheral blood of female Balb/c mice following exposure to per os lead acetate trihydrate or plain drinking water for two weeks and after a two-week recovery period. Data sets were RMA-normalized and dose-specific signatures were generated using established methods of supervised classification and binary regression. Pathway activity was analyzed using the ScoreSignatures module from GenePattern. Conclusions/Significance: The low-level Pb signature was 93% sensitive and 100% specific in classifying samples a leaveone-out crossvalidation. The high-level Pb signature demonstrated 100% sensitivity and specificity in the leave-one-out crossvalidation. These two signatures exhibited dose-specificity in their ability to predict Pb exposure and had little overlap in terms of constituent genes. The signatures also seemed to reflect current levels of Pb exposure rather than past exposure. Finally, the two doses showed differential activation of cellular pathways. Low-level Pb exposure increased activity of the interferon-gamma pathway, whereas high-level Pb exposure increased activity of the E2F1 pathway.

Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 3859 Hematopoietic stem cell (HSC) regeneration is influenced by specialized bone marrow (BM) microenvironments, but the mechanisms that drive HSC regeneration remain incompletely defined. We have recently reported that deletion of the pro-apoptotic proteins, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs)(Tie2Cre;Bak-/-;BaxFl/- mice) caused a significant protection of the BM HSC pool and the BM sinusoidal vasculature in mice following high dose total body irradiation (TBI). We also confirmed that this protection of the BM HSC pool was caused by protection of BM Tie2+ ECs via generation of chimeric mice (Tie2Cre;Bak-/-;BaxFl/- BM; wild type BM ECs) which contained 4.8-fold less BM long-term repopulating HSCs compared to mice bearing deletion of Bak and Bax in both BM HSCs and BM ECs. In order to determine the mechanism through which Tie2+ BM ECs regulate HSC regeneration, we generated primary BM EC lines from Tie2Cre;Bak-/-;BaxFl/- mice and Tie2Cre;Bak-/-;BaxFl/+ control mice. We then compared the capacity for Bak/Bax -/- BM ECs to support BM HSC regeneration in vitro compared to Bak/Bax +/− BM ECs. BM c-kit+sca-1+lin- (KSL) stem/progenitor cells were irradiated with 300 cGy and then placed in 7 day culture with Bak/Bax -/- BM ECs or Bak/Bax +/− BM ECs. Culture with Bak/Bax -/- BM ECs did not yield a significant increase in total viable cells, but yielded 2000-fold increased number of BM KSL cells (p < 0.05, n=3) compared to cultures with Bak/Bax +/− ECs. This significant expansion of phenotypic BM stem/progenitor cells corresponded to a 4-fold increase in CFU-S12 cells in the Bak/Bax -/- EC cultures vs. Bak/Bax +/− EC cultures (p=0.01, n=5). We subsequently compared the level of expression of several microenvironmental ligands which are putatively involved in regulating hematopoiesis. We found that BM ECs from Tie2Cre;Bak-/-;BaxFl/- mice had 37-fold lower expression of stromal-derived factor-1 (SDF-1, CXCL12) compared to BM ECs from Tie2Cre;Bak-/-;BaxFl/+ mice. Moreover, 7 days after TBI, Tie2Cre;Bak-/-;BaxFl/- mice had a 41-fold increase in total viable BM cell counts and had a persistently lower SDF-1 expression on BM ECs (2.7-fold) compared to Tie2Cre;Bak-/-;BaxFl/+ mice (p=0.003). Therefore, we hypothesized that inhibition of SDF-1 signaling might facilitate hematopoietic regeneration following injury. Interestingly, the addition of a blocking anti-SDF1 antibody to cultures of irradiated BM KSL cells with Bak/Bax -/- ECs caused a 50% increase in total cell recovery (p Disclosures: No relevant conflicts of interest to declare.

Abstract 3734 We have recently demonstrated that targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs) causes the protection of the BM sinusoidal vasculature, and the BM hematopoietic stem cell (BM HSC) pool following high dose total body irradiation (TBI). Since Tie2 is expressed on BM ECs and a small subset of quiescent BM HSCs, we developed a complementary model utilizing VE-cadherin Cre mice to more specifically confirm the function of BM ECs in regulating hematopoietic reconstitution in vivo. VE-cadherinCre;Bak−/−;BaxFl/− mice, which bear deletion of Bak and Bax in VE-cadherin+ ECs, and VE-cadherinCre;Bak−/−;BaxFl/+ control mice were generated and we compared the hematopoietic responses of these animals to high dose TBI. At steady state, these mice showed no difference in total BM cells, bone marrow ckit+sca+lineage- (KSL) progenitor cells, BM colony forming cells (CFCs), or BM colony forming unit spleen day 12 (CFU-S12) counts. At 2 hours after exposure to 500cGy TBI, the mice also did not demonstrate any significant differences in total BM cells, BM KSL cells, CFCs, or CFU-S12. However, 7 days after exposure to 500cGy TBI, VE-cadherin;Bak−/−;BaxFl/− mice displayed a 2-fold increase in total BM cells (p=0.006), a 3-fold increase in BM CFCs (p=0.0009), and 4-fold increase in BM CFU-S12 (p=0.079) compared to VE-cadherinCre;Bak−/−;BaxFl/+ control mice. Microscopic examination confirmed severe hypocellularity and corruption of the BM sinusoidal vasculature at day +7 post-TBI in VE-cadherinCre;Bak−/−;BaxFl/+ mice, whereas VE-cadherinCre;Bak−/−;BaxFl/− mice displayed nearly normal cellularity and preserved BM sinusoidal vessels. Taken together, these data demonstrate that VE-cadherin+ BM ECs regulate hematopoietic reconstitution in vivo and that targeted therapies aimed at augmentation of BM EC function can accelerate hematopoietic regeneration in vivo. Currently, we are comparing the cytokine production and gene expression profiles of VE-cadherinCre;Bak−/−;BaxFl/− BM ECs versus VE-cadherinCre;Bak−/−;BaxFl/+ BM ECs to identify candidate BM EC-derived proteins which are responsible for the protection of the BM stem/progenitor cell pool from radiation injury. VE-cadherin+ BM ECs represent an attractive mechanistic target for the identification of signaling pathways that regulate hematopoietic regeneration following injury. Disclosures: Chao: Genzyme: Research Funding.

Abstract 1498 Poster Board I-521 Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 248 Radiation and chemotherapy cause myelosuppression in patients via damage to bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells. It has been shown that BM HSCs reside in close association with osteoblasts and that BM osteoblasts regulate the maintenance of quiescent HSCs in vivo. HSCs also reside in proximity to BM sinusoidal vessels but the function of BM endothelial cells (BM ECs) in regulating HSC fate in vivo remains less well understood. We hypothesized that BM ECs play a critical role in regulating BM hematopoietic reconstitution following stress. In order to test this hypothesis, we utilized Cre-LoxP recombination to generate mice bearing targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ BM ECs (Tie2Cre;Bak−/− ;BaxFl/− mice) and measured their hematopoietic response to total body irradiation (TBI). Tie2Cre;Bak−/−;BaxFl/− mice (EC-Bak;Bax knockouts) were compared with Tie2Cre;Bak−/−;BaxFl/+ mice (EC-Bak;Bax (+) mice) which have constitutive Bak deletion but retain Bax in Tie2+ BM ECs. EC-Bak;Bax knockout mice had no detectable Bak or Bax by Western Blot in vascular tissues. After exposure to 100 cGy total body irradiation (TBI), EC-Bak;Bax knockout mice displayed a 2-fold increase in total viable BM cells (p=0.04), a 3-fold increase in BM ckit+sca+lineage- (KSL) progenitor cells, a 3-fold increase in colony-forming unit-spleen day 12 (CFU-S12) content (p=0.0003) and a 2-fold increase in 4 week competitive repopulating units (CRUs) compared to EC-Bak;Bax (+) mice. After 300 cGy TBI, comparable radioprotection was observed in the EC-Bak;Bax knockout mice, with a 1.9-fold increase in total BM cells, a 2.4-fold increase in CFU-S (p=0.01) and a 3.6-fold increase in 4 week CRU compared to EC-Bak;Bax (+) mice. Taken together, these results suggested that targeted protection of Tie2+ BM ECs from the intrinsic pathway of apoptosis mediated protection of BM hematopoietic stem/progenitor cells following TBI. Since Tie2 is expressed by BM ECs and a small subset of quiescent BM HSCs, we carried out experiments to determine whether the radioprotection we observed in EC-Bak;Bax knockout mice was caused autonomously by protection of Tie2+ BM ECs or Tie2+ HSCs. We transplanted 4 × 106 BM cells from EC-Bak;Bax knockout mice into lethally irradiated (950 cGy) wild type (WT) B6.SJL mice such that, after 12 weeks post-transplant, the recipient mice were chimeric for Bak and Bax deletions only in hematopoietic cells while retaining a wild type BM microenvironment (HSC-Bak;Bax knockout;EC-wild type), verified by qRTPCR. At 16 weeks post-transplant, the chimeric recipient mice were then exposed to 100 and 300 cGy TBI and we compared the hematopoietic response of these mice to that of EC-Bak;Bax knockout mice. Interestingly, after 100 cGy TBI, the hematopoietic response of the chimeric mice was comparable to that of wild type C57Bl6 mice and revealed a significant reduction in total viable BM cells (1.3-fold), BM KSL cells (7.4 fold) and BM CRU-4 weeks (9-fold) compared to 100 cGy-irradiated EC-Bak;Bax knockout mice. Significant reductions in total BM cells and BM CRU content were also observed in chimeric mice compared to EC-Bak;Bax knockout mice following 300 cGy TBI. These results suggest that deletion of Bak and Bax-mediated apoptosis in Tie2+ BM ECs protects BM hematopoietic stem and progenitor cells from ionizing radiation damage and this protection is largely autonomous to Tie2+ BM ECs. More generally, these data demonstrate that protection of BM ECs from the deleterious effects of ionizing radiation results in augmented hematopoietic reconstitution following TBI. This study demonstrates that Tie2+ BM ECs have an essential role in regulating hematopoietic regeneration following injury and reveals that BM ECs are a novel and attractive therapeutic target to augment hematopoietic reconstitution in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 1247 Poster Board I-269 Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal, mature B-cell lymphocytosis, lymphadenopathy and splenomegaly. The pathogenesis of CLL is thought to be related to abnormal programmed cell death mechanisms but the precise contribution of several pro-apoptotic genes remains unknown. Elucidation of the pathogenesis of CLL is also limited by the lack of animal models which reproduce the CLL phenotype. We present a transgenic mouse model in which conditional, tissue-specific deletion of Bax is achieved in Tie2+ hematopoietic stem cells (HSCs) via Cre-LoxP recombination. In this model, Tie2Cre;Bak-/-;BaxFl/- mice carried constitutive deletion of Bak and targeted deletion of Bax in Tie2+ BM HSCs. Surprisingly, the combined deletion of Bak and Bax in Tie2+ cells caused a pronounced leukocytosis with total WBCs>100,000 in 8-week old mice. In contrast, Tie2Cre;Bak-/-;BaxFl/+ mice, which retained one Bax allele, had normal peripheral blood WBCs comparable to wild type C57Bl6 mice (range 4-10,000;p Disclosures No relevant conflicts of interest to declare.

The ability to undergo self-renewal is a defining feature of hematopoietic stem cells (HSCs) but the extrinsic signals which regulate HSC self-renewal remain unclear. We performed a genome-wide expression analysis on primary human brain ECs (HUBECs, n=10) which support the ex vivo expansion of HSCs in non-contact culture (Blood100:4433–4439; Blood105:576–583) and non-brain ECs which do not support HSC expansion (n=8) in order to identify soluble proteins overexpressed by the HSC-supportive HUBECs. We identified pleiotrophin (PTN), an 18 kD heparin binding growth factor, to be 32-fold overexpressed in HUBECs as compared to non-supportive EC lines. PTN has established activity in angiogenesis, embryogenesis, neuronal cell growth and tumorigenesis, but has no known function in hematopoiesis. We first tested whether secreted PTN was responsible for the amplification of HSCs that we have observed in co-cultures of HSCs with HUBECs via “loss of function” studies in which a blocking anti-PTN antibody was added to HUBEC cultures and HSC content was measured. Competitive repopulating unit (CRU) assays were performed in which limiting doses of donor CD45.1+ bone marrow (BM) 34−c-kit+sca-1+lin− (34-KSL) HSCs (10, 30 or 100 cells) or their progeny following 7 day non-contact culture with HUBECs + IgG or HUBECs + a blocking anti-PTN were transplanted into lethally irradiated CD45.2+ C57Bl6 mice. Mice transplanted with the progeny of 34-KSL cells cultured with HUBECs demonstrated 4–6 fold increased levels of donor-derived CD45.1+ multilineage repopulation at 8-, 12- and 24-weeks post-transplantation as compared to mice transplanted with input 34-KSL cells. In contrast, mice transplanted with the progeny of 34-KSL cells following culture with HUBECs + anti-PTN demonstrated significant reduction in donor CD45.1+ cell repopulation compared to mice transplanted with the progeny of HUBEC cultures and no difference in donor CD45.1+ cell engraftment compared to mice transplanted with input 34-KSL cells. CRU frequency within day 0 34-KSL cells was estimated to be 1 in 40 cells (95% Confidence Interval [CI]: 1/22-1/72), whereas the CRU estimate within the progeny of 34-KSL cells following HUBEC culture was 1 in 4 cells (CI: 1/2-1/9). The addition of anti-PTN to the HUBEC co-culture decreased the CRU estimate to 1 in 29 cells (CI: 1/16-1/52), suggesting that PTN signaling was responsible for the expansion of HSCs observed in HUBEC co-cultures. In order to confirm whether PTN is indeed a novel growth and self-renewal factor for HSCs, we next performed “gain of function” studies in which 34-KSL cells were placed in liquid suspension cultures with cytokines (thrombopoietin 50 ng/mL, SCF 120 ng/mL, flt-3 ligand 20 ng/mL) with and without the addition of increasing doses of recombinant murine PTN (10, 50 and 100 ng/mL) and total cell expansion and HSC content were compared. The addition of 100 ng/mL PTN to cytokine cultures caused a 20-fold increase in KSL cell content at day 7 compared to input (P