Lineage-negative Bone Marrow Cells Research Articles

SRSF2 Is Essential For Hematopoiesis and Its Mutations Dysregulate Alternative RNA Splicing In MDS

Myelodysplastic syndromes (MDS) are a group of neoplasms that are ineffective in generating multiple lineages of myeloid cells and have various risks to progress to acute myeloid leukemia. Recent genome-wide sequencing studies reveal that mutations in genes of splicing factors are commonly associated with MDS. However, the importance of these splicing factors in hematopoiesis has been unclear and the causal effect of their mutations on MDS development remains to be determined. One of these newly identified genes is SRSF2, and its mutations have been linked to poor survival among MDS patients. Interestingly, most of SRSF2 mutations occur at proline 95 and the majority of these mutations change this proline to histidine (P95H). Given that SRSF2 is a well-characterized splicing factor involved in both constitutive and regulated splicing, we hypothesize that SRSF2 plays an important role in normal hematopoiesis and the SRSF2 mutations induce specific changes in alternative splicing that favor disease progression. We first examined the role of SRSF2 in hematopoiesis by generating Srsf2 null mutation in mouse blood cells via crossing conditional Srsf2 knockout mice (Srsf2f/f) with blood cell-specific Cre transgenic mice (Vav-Cre). The mutant mice produced significantly fewer definitive blood cells (10% of wild type controls), exhibited increased apoptosis in the remaining blood cells, and died during embryonic development. Importantly, we detected no hematopoietic stem/progenitor cells (lineage-/cKit+) in E14 fetal livers of Vav-Cre/Srsf2f/f mice. These results indicate that SRSF2 is essential for hematopoiesis during embryonic development. We next examined the role of SRSF2 in adult hematopoiesis by injecting polyIC into mice that carry a polyIC inducible Cre expression unit. Unexpectedly, after multiple polyIC treatments, the Srsf2f/f mice stayed alive during several months of observation. Time course genotyping analyses of polyIC treated mice revealed an increased rate of incomplete Srsf2 deletion in peripheral blood cells. These observations suggest that Srsf2 ablation did not cause immediate cell lethality in differentiated blood cells, but the gene is indispensable for the function of blood stem/progenitor cells. Since mutations of splicing factors are generally heterozygous in MDS patients, we also examined mice with Srsf2+/- blood cells. No obvious defect of hematopoiesis was observed under normal conditions or in response to stress with 5-FU treatment and sublethal irradiation. To gain molecular insight into the splicing activity of MDS-associated mutant forms of SRSF2, we performed large-scale alternative splicing surveys by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) previously developed in our lab, which offers a robust and cost-effective platform for splicing profiling. Compared to vector transduction controls, we found that overexpression of both wild type and P95H SRSF2 induced many, but distinct changes in alternative splicing in lineage-negative bone marrow cells, and importantly, we noted several changes in genes with known roles in hematopoietic malignancies that were uniquely induced by the mutant SRSF2. To further link the mutations to altered splicing in MDS patients, we also applied RASL-seq to a large number of MDS patient samples with or without mutations in SRSF2 or other splicing regulators. The data revealed a specific set of alternative splicing events that are commonly linked to MDS with splicing factor mutations. These findings strongly suggest that many of these mutations in splicing regulators are gain-of-function mutations that are causal to MDS. In conclusion, we report that SRSF2 plays an essential role in hematopoietic stem/progenitor cells and that the MDS-associated mutations in SRSF2 have a dominant effect on RNA alternative splicing. These findings provide functional information and molecular basis of SRSF2 and its MDS-related mutations in hematopoiesis and related clinical disorders. Disclosures:No relevant conflicts of interest to declare.

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

BackgroundThe WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/ResultsWe obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. ConclusionOncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. [Display omitted] ▪ Disclosures:No relevant conflicts of interest to declare.

Stress Response Transcription Factor Egr-1 As Tumor Suppressor Of CML

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for different leukemias. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. Although the Tyrosine Kinase Inhibitor (TKI) imatinib mesylate (Gleevec, Novartis) is effectively used on CML patients, resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. It was observed that Egr-1 expression is reduced in bone marrow (BM) of CML patients, and its expression is further reduced in more advanced stages of CML. Consistent with this data, Egr-1 expression is reduced in BCR-ABL-expressing murine BM. The tumor suppressor role of Egr-1 in CML was validated using mouse models. Lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, no statistically significant difference in the percentage of stem cells (Lin-Sca+c-Kit+, LSK) was observed between Egr-1 WT and Egr-1-/- BM. Thus, the BM stem cell compartment of the Egr-1-/- mice does not offer a quantitative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Data from serial colony transfer and studies using spleens from diseased mice as well as BCR-ABL-expressing BM will be presented. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression, that can be used to treat CML, as well as other leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 4585: Breaking metastatic dormancy during surgical resection of a primary tumor and implications for treatment strategies.

Abstract At the time of a cancer diagnosis, most patients have localized tumors. Despite elaborate staging schemas for each cancer type in an attempt to stratify patients, the vast majority of patients that die will do so from metastatic disease. We hypothesized that surgery augments the already ongoing activation and mobilization of bone marrow-derived progenitor cells that are critical to colonizing tumor cells at distant sites. These bone marrow-derived cells, by inducing a local inflamed tumor microenvironment, provide survival signals to these seeding tumor cells. Our data show increased metastatic burden in the lung after surgical resection of the primary tumor using two murine cancer models, B16 melanoma and E0771 breast carcinoma. In these models, we also show a surge in hematopoietic and endothelial progenitor cells in the hours and days immediately following resection of the primary tumor, which is not similarly observed in control mice, where surgery was performed in the absence of the primary tumor. We also confirmed that a factor specific to the plasma of the tumor-bearing mice is responsible for this mobilization by using in vitro migration assays, whereby plasma from tumor-bearing mice and surgically resected mice induced an increased migration of lineage negative bone marrow cells compared to the plasma of wild type mice. We confirmed increased levels of MCP-1 and MCSF, both known to mobilize progenitor cells, in the plasma of mice with surgical resection. Additionally, targeting these bone marrow derived hematopoietic and endothelial progenitor cells with Pazopanib prevents the surge in bone marrow-derived cells into the circulation, abolishes the enhanced metastatic spread in mice undergoing surgical resection of the primary tumor, and provides a significant prolongation of survival. Finally, we correlated these data to a cohort of breast cancer patients where circulating levels of progenitor cells were analyzed at time points before and after surgery, which confirmed the mobilization of progenitor cells with surgery. Together, these results provide evidence for the increased risk of metastatic spread after surgical resection of the primary tumor and suggest that blocking progenitor cell mobilization by adjuvant treatment during or immediately following surgery, the incidence of metastatic recurrence may be reduced. Citation Format: Selena R. Granitto, Amber Giles, Simon Lavotshkin, Daniel Rutigliano, David Lyden, Rosandra N. Kaplan. Breaking metastatic dormancy during surgical resection of a primary tumor and implications for treatment strategies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4585. doi:10.1158/1538-7445.AM2013-4585

Next Gen Dissection of Gfi1 Dependent Transcriptome in Myeloid Progenitors Reveals Global Control of Multiple Transcriptional Programs Including Coding and Non Coding RNAs

AbstractAbstract 111Growth Factor Independence 1 (Gfi1) is a regulator of HSC maintenance, and lack of Gfi1 leads to myeloid progenitor accumulation and neutropenia. The Gfi1 transcription factor contains both a SNAG repressor domain, and a zinc-finger DNA-binding domain. A proline to alanine substitution at position 2 in the SNAG domain (P2A) blocks RCoR/Lysine Specific Demethylase 1 (LSD1) interaction and abrogates Gfi1-mediated repression. Mouse models have been developed which either delete Gfi1 exons 2–3 (Gfi1Δex2-3) or knock in the P2A mutation (GfiP2A). Both models are neutropenic, and demonstrate an accumulation of arrested myeloid progenitors. Transgenic shRNA knockdown of Lsd1 in the hematopoietic system results in the accumulation of myeloid progenitors and inhibits terminal granulocytic differentiation; however, the role of Lsd1/Gfi1 in this process has not been established. To characterize the global role of Gfi1 dependent transcriptional regulation in progenitor maintenance and lineage determination, we utilized a Next Generation Sequencing (NGS) approach to profile Gfi1Δex2-3and GfiP2A lineage negative bone marrow cells. Using gene-level analyses, a representative group of 1,019 genes were identified that exhibited more than 1.5 fold deregulation (by FPKM) and differential gene expression relative to WT sample profiles in at least one model. Interestingly, some of the Gfi1-dependent genes encode putative lncRNAs, which were validated independently. Using an shRNA mediated approach, we identified one lncRNA whose expression (like Gfi1) affects the granulo- monocytic balance of differentiating myeloid progenitors. Bioinformatic interrogation revealed that genes regulated by Gfi1 are mainly involved in normal and aberrant progenitor regulation, with processes such as immunology, signal transduction, and myelopoiesis modestly represented. Given the phenotype of Lsd1 knockdown in hematopoietic cells, we hypothesized that loss of Gfi1 (and/or SNAG) activity would disrupt Lsd1 activity at Gfi1-target genes, resulting in the accumulation of histone 3 lysine 4 dimethyl (H3K4me2) marks. For the subset of genes deregulated in both Gfi1Δex2-3and GfiP2A models, chromatin immunoprecipitation revealed direct Gfi1 DNA binding and increased H3K4me2 marks in a majority of the significantly deregulated genes. We previously showed that accumulation of Gfi1 null myeloid progenitors was dose dependent upon the Gfi1 target gene HoxA9. In our current experiments, HoxA9 was deregulated in both Gfi1 null and Lsd1 knockdown, bound by Gfi1 in ChIP, and showed increased H3K4me2 peaks upon Gfi1 loss; providing a mechanistic explanation for the common phenotypes of Gfi1 null and Lsd1 knockdown in hematopoietic progenitors. Our results collectively reveal multiple transcriptional circuits controlled by the Gfi1/Lsd1 complex underlying normal progenitor maintenance and differentiation. Disclosures:No relevant conflicts of interest to declare.

Genistein Protects Hematopoietic Stem Cells Against G-CSF Induced DNA Damage

Abstract 3486Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. A particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. Our results suggest that genistein’s effects are mediated primarily through inhibition of HSC proliferation. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, we demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support. Disclosures:No relevant conflicts of interest to declare.

Platelets: An Efficient Depot for Generation and Distribution of Lysosomal Idua in Enzyme-Deficient MPS I Mice.

Abstract 3157Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)-mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Platelets are terminally differentiated, enucleated cells full of secretory granules. Restricting transgene expression to platelets may reduce the risk for activating oncogenes in HSCs and most of their progeny, and take advantage of their rapid turnover and professional secretion function. In this study, we evaluated the feasibility of employing megakaryocyte-platelet as a depot for the over-expression and release of alpha-L-iduronidase (IDUA), the lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). Utilizing a human megakaryocytic DAMI cell line, we found that a hybrid human ankyrin-1 promoter (K) containing ALAS2 intron 8 enhancer and HS40 core element from human alpha LCR could introduce robust intracellular IDUA expression (over 500-fold of background) and significant enzyme release (120-fold). Upon introduction to megakaryocytic differentiation as verified by FACS analysis with PI staining and morphologic evaluation, the elevated intracellular IDUA levels remained unchanged, while released IDUA increased steadily by 30-fold. We then evaluated in vivo IDUA production/release from platelets in an enzyme-deficient MPS I mouse model. Lineage-negative bone marrow (BM) cells were isolated from MPS I mice using immunomagnetic cell sorting, and transduced twice with LV-K-IDUA-ires-GFP (KIiG) for a total multiplicity of infection at 30. Five-months after 1° transplantation, GFP+ platelets were detected by FACS analysis with CD41-PE staining and observed by Image X analysis. To generate animals with various transgene-dosages, 2° transplantation was performed into MPS I mice using a serial dilution of BM from 1° MPS/KIiG (51–65% transduction efficiency) with BM of MPS I. FACS analysis revealed that the percentage of GFP+ platelets was correlated directly to transduction efficiencies in 2° recipients as determined by real-time qPCR of BM 5 months post transplantation. Moreover, the intra-platelet IDUA enzyme activities were associated linearly with the changes of GFP+% platelets (r2=0.95) among MPS I mice with different transgene-dosages, suggesting that the lysosomal IDUA from transgene overexpression could be sorted and packaged into platelets with proper catalytic function. Noticeably 1% gene transfer efficiency as shown by 1% GFP+ platelets was sufficient to introduce the IDUA levels comparable to those found in wild-type platelets. To determine if platelet-derived IDUA could be released, we conducted Ca-induced platelet activation as verified by FACS analysis with Annexin V-APC and CD41-PE staining, resulting in linear correlation (r2=0.98) of released IDUA with intra-platelet IDUA. Interestingly, the plasma IDUA levels were also linearly correlated with GFP+% platelets (r2=0.96) among MPS I with different transgene-dosages. Competitive uptake assay using lymphoblastoid cells derived from a MPS I patient (LCLmps) demonstrated steady increase of intracellular IDUA with increasing amounts of extracellular enzyme levels, which was blocked by the presence of mannose-6-phosphate (M6P). This suggested that platelet-released IDUA retained its intercellular trafficking capability via M6P receptor. Finally normalization of aberrant lysosomal morphology was observed in LCLmps exposed to platelet-released IDUA as determined by in situ immunostaining with LysoTracker, indicating functional proficiency of platelet-derived IDUA to cross-correct deficit in patient’s cells. These results demonstrate for the first time that megakaryocytes/platelets are capable of over-producing, packaging and storing a lysosomal enzyme which retains proper catalytic activity, lysosomal enzyme trafficking and endogenous M6PR-mediated uptake, as well as the ability of cross-correction in patient’s cells. This data warrants further evaluation for the potential application of megakaryocytes/platelets in treating lysosomal storage diseases, especially for those, such as Gaucher disease, when the desirable enzyme is sensitive to neutral pH in serum and protection of enzyme in platelets may provide continuous, real-time enzyme release by low physiological levels of platelet activation. Disclosures:No relevant conflicts of interest to declare.

A Specific DNA Methylation Pattern Is Associated with Delayed Leukemogenesis and Altered Response to Chemotherapeutic Treatment in an Inducible DNMT3b Expressing Mouse Model

AbstractAbstract 398DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT3B overexpression on leukemogenesis using a tetracycline-inducible DNMT3B mouse model. To analyze the impact of DNMT3B overexpression on leukemogenesis, we retrovirally co- transduced lineage-negative bone marrow cells of wildtype and DNMT3B transgenic mice with an MSCV- cMyc-bcl2 or MSCV-MLL-AF9-hCD4 and an MSCV- tTA- GFP expressing vector. Using these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. To verify that expression of DNMT3b in this mouse model was able to induce DNA methylation, we performed Reduced Representation Bisulfite Sequencing (RRBS) on cMyc-Bcl2 leukemic bone marrow cells. Analysis of the global methylation levels revealed a strong hypermethylation in DNMT3b overexpressing bone marrow cells. Differentially methylated regions (DMR) were defined as those regions with at least 30% difference in methylation levels. DMRs were mainly located in promoter regions and first introns and the heatmap of these DMRs confirms distinct hypermethylation in DNMT3b transgenic cells whereas very few regions become hypomethylated in transgenic cells when compared to wildtype cells. This specific methylation pattern could be responsible for altered gene expression and consecutively altered (stem) cell characteristics.To analyze the influence of changed DNA methylation levels induced by expression of DNMT3b on leukemogenesis, sorted GFP+cells were transplanted into sublethally irradiated wildtype recipients. Mice with and without DNMT3B expression developed leukemia with splenomegaly and a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. Mice with cMyc-Bcl2- and MLL-AF9-induced leukemia showed a clear myeloid phenotype with enhanced expression of the stem cell marker c-kit on MLL-AF9 leukemic cells. To consider the leukemogenic capacity, we transplanted the leukemic blasts into secondary recipients. cMyc-Bcl2 induced leukemia of both, wildtype and DNMT3b recipients was transplantable and lethal. DNMT3B tg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of cMyc-Bcl2 induced leukemic DNMT3B cells died significantly later (p= 0.04).As differential methylation can also have an effect on the responsiveness to chemotherapy, spleen cells from MLL-AF9 leukemic mice were treated with different cytotoxic and demethylating drugs to see if DNMT3b induced hypermethylation confers a resistance or increased sensitivity to chemotherapeutic treatment. DNMT3b overexpressing cells were more sensitive to treatment with Azacytidine and Decitabine.Taken together, these findings demonstrate that expression of DNMT3b in a doxycycline-regulatable mouse model induces widespread DNA hypermethylation, whereas a few small regions become hypomethylated when compared to wildtype cells. This specific methylation pattern is associated with delayed leukemogenesis and DNMT3b expression significantly impaired leukemia maintenance. In addition, response to chemotherapeutic treatment is altered by DNMT3b induced hypermethylation which underlines the importance of epigenetic regulatory mechanisms as promising targets for new therapeutic strategies in AML. Disclosures:Müller-Tidow:Celgene: Lecture fees Other; Deutsche Forschungsgemeinschaft: Research Funding.

MicroRNA-10A* and MicroRNA-21 Modulate Endothelial Progenitor Cell Senescence Via Suppressing High-Mobility Group A2

Endothelial progenitor cells (EPCs) contribute to the regeneration of endothelium. Aging-associated senescence results in reduced number and function of EPCs, potentially contributing to increased cardiac risk, reduced angiogenic capacity, and impaired cardiac repair effectiveness. The mechanisms underlying EPC senescence are unknown. Increasing evidence supports the role of microRNAs in regulating cellular senescence. We aimed to determine whether microRNAs regulated EPC senescence and, if so, what the underlying mechanisms are. To map the microRNA/gene expression signatures of EPC senescence, we performed microRNA profiling and microarray analysis in lineage-negative bone marrow cells from young and aged wild-type and apolipoprotein E-deficient mice. We identified 2 microRNAs, microRNA-10A* (miR-10A*), and miR-21, and their common target gene Hmga2 as critical regulators for EPC senescence. Overexpression of miR-10A* and miR-21 in young EPCs suppressed Hmga2 expression, caused EPC senescence, as evidenced by senescence-associated β-galactosidase upregulation, decreased self-renewal potential, increased p16(Ink4a)/p19(Arf) expression, and resulted in impaired EPC angiogenesis in vitro and in vivo, resembling EPCs derived from aged mice. In contrast, suppression of miR-10A* and miR-21 in aged EPCs increased Hmga2 expression, rejuvenated EPCs, resulting in decreased senescence-associated β-galactosidase expression, increased self-renewal potential, decreased p16(Ink4a)/p19(Arf) expression, and improved EPC angiogenesis in vitro and in vivo. Importantly, these phenotypic changes were rescued by miRNA-resistant Hmga2 cDNA overexpression. miR-10A* and miR-21 regulate EPC senescence via suppressing Hmga2 expression and modulation of microRNAs may represent a potential therapeutic intervention in improving EPC-mediated angiogenesis and vascular repair.

Abstract 16895: Micrornas Control Endothelial Progenitor Senescence via Regulating Plk2 Expression

Background: With advancing age the regenerative properties of somatic stem cells deteriorate in part due to cellular senescence, eventually leading to the diminished repair capability. Endothelial progenitor cells (EPCs) play an important role in angiogenesis. Several miRNAs have been reported to be critical modulators for EPC plasticity. The aim of this study was to determine whether miRNAs regulated age-related senescence in lineage negative bone marrow cells (lin - BMCs) enriched for EPCs. Methods: Lin - BMCs were isolated from young (3-week-old) wild type (WT) and apoE -/- C57BL/6 mice and aged WT (29-month-old) and apoE -/- (12 month-old) mice. Global miRNA and gene expression profiling were analyzed. Results: Using genome-wide miRNA analysis, we found that miR-146a was increased 6.3-fold and 3.4-fold in aged WT and apoE -/- lin - BM cells, respectively, relative to their young counterparts. Microarray analysis showed that the Polo-like kinase 2 (Plk2), a member of the ‘polo’ family of serine/threonine protein kinases that are critical regulators of cell cycle progression or apoptosis, was decreased 18-fold in aged lin - BMC as compared to young cells. Luciferase assay showed miR-146a substantially inhibited WT Plk2-3’-UTR-luciferase activity in 293T cells, but not mutant Plk2-3’-UTR-luciferase reporter. Overexpression of lentiviral-derived miR-146a resulted in reduction of Plk2 expression, increased senescence-associated (SA) β-gal expression and decreased vascular tube formation in young EPCs, whereas miR-146a antagomir rejuvenated aged EPCs, causing increased Plk2 expression, decreased SA β-gal expression and enhanced vascular tube formation. Furthermore, knockdown of Plk2 by its siRNA resulted in increased senescence and diminished vascular tube formation. Conclusions: Using genomic and functional studies, we have discovered miR-146a controls age-associated EPC senescence via regulating Plk2 expression, thus negatively affecting their vascular repair capacity, which may impact atherogenesis. These findings advance our understanding of EPC aging and provide molecular targets for genetic modification to increase the therapeutic efficacy of EPCs in preventing and repairing vascular damage in atherosclerosis.

MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Abstract 551Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

DNMT3B Expression Delays Leukemogenesis,

Abstract 3446Acute myeloid leukaemia (AML) is a malignant disease with poor prognosis, which is, among other biological features, characterized by epigenetic changes including alterations in DNA methylation. DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. We analyzed the effects of DNMT overexpression on leukemogenesis using an inducible DNMT3B mouse model (Linhart et al., 2007). To analyse the impact of DNMT3B overexpression on leukemia we retrovirally co-transduced lineage-negative bone marrow cells of wildtype and DNMT3Btg mice with a MSCV-cMyc-bcl2 and a MSCV-tTA-GFP containing vector. Under these conditions, doxycycline suppressed DNMT3B expression whereas absence of doxycycline led to overexpression of DNMT3B on the mRNA and protein level. DNMT3B overexpression was not toxic since colony formation in vitro did not differ between DNMT3B expressing and physiologically expressing cells. To analyze leukemogenesis, 5 × 104 sorted GFP-positive cells were transplanted into sublethally irradiated wildtype recipients. Both recipients of transduced wildtype cells and recipients of transduced DNMT3Btg cells developed leukemia with a tendency of delayed leukemogenesis in DNMT3B overexpressing mice. GFP positive leukemic cells were sorted and doxycycline regulated DNMT3B expression was verified by Western blot analysis in vitro. To determine the repopulation capacity of the leukemic cells we performed transplantation of GFP-positive primary leukemia cells into secondary wildtype recipients. Leukemia of both, wildtype and DNMT3B-overexpressing donors was transplantable and lethal. However, DNMT3Btg leukemic cells were severely impaired in leukemia development in secondary recipients. Secondary recipients of leukemic DNMT3Btg cells died significantly later (p= 0.02). Taken together, these findings demonstrate that DNMT3B expression impairs leukemia maintenance. Loss of DNMT activity might contribute to the pool size of leukemia initiating cells. Disclosures:Krug:Boehringer Ingelheim: Research Funding.

Notch signaling regulates the development of a novel type of Thy1‐expressing dendritic cell in the thymus

Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8α(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs.

Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells

AbstractAbstract 878Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures:No relevant conflicts of interest to declare.

Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells

HOXB4, a member of the Homeobox transcription factor family, promotes expansion of hematopoietic stem cells and hematopoietic progenitor cells in vivo and ex vivo when overexpressed. However, the molecular mechanisms underlying this effect are not well understood. To identify direct target genes of HOXB4 in primary murine hematopoietic progenitor cells, we induced HOXB4 function in lineage-negative murine bone marrow cells, using a tamoxifen-inducible HOXB4-ER(T2) fusion protein. Using expression microarrays, 77 probe sets were identified with differentially changed expression in early response to HOXB4 induction. Among them, we show that Hemogen (Hemgn), encoding a hematopoietic-specific nuclear protein of unknown function, is a direct transcriptional target of HOXB4. We show that HOXB4 binds to the promoter region of Hemgn both ex vivo and in vivo. When we overexpressed Hemgn in bone marrow cells, we observed that Hemgn promoted cellular expansion in liquid cultures and increased self-renewal of myeloid colony-forming units in culture, partially recapitulating the effect of HOXB4 overexpression. Furthermore, down-regulation of Hemgn using an shRNA strategy proved that Hemgn contributes to HOXB4-mediated expansion in our myeloid progenitor assays. Our results identify a functionally relevant, direct transcriptional target of HOXB4 and identify other target genes that may also participate in the HOXB4 genetic network.

Tumor Necrosis Factor Receptors Support Murine Hematopoietic Progenitor Function in the Early Stages of Engraftment

Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.

In vitro differentiation of lineage‐negative bone marrow cells into microglia‐like cells

Microglia are believed to be the only resident immune cells in the CNS, originating from hematopoietic-derived myeloid cells and invading the CNS during development. However, the detailed mechanisms of differentiation and transformation of microglial cells are not fully understood. Here, we demonstrate that murine microglial cells show two morphological forms in vitro, namely, small round cells expressing CD11b, Iba1, triggering receptor expressing on myeloid cells-2 (TREM2), and weakly expressing major histocompatibility complex class II and large flat cells expressing only CD11b and Iba1. Moreover, lineage-negative bone marrow (LN) cells cultured with primary mixed glial culture cells could differentiate into only the small round microglia-like cells, despite the absence of CCR2 and Gr-1 expression. Addition of macrophage colony stimulating factor (M-CSF) to LN cell culture allowed the proliferation and expression of TREM2 in LN cells, and the addition of neutralizing anti-M-CSF antibodies suppressed the proliferation of LN cells despite the expression of TREM2. When LN cells were cultured with M-CSF, the number of small round cells in the culture was considerably low, indicating that the small round morphology of the immature cells is not maintained in the presence of only M-CSF. On the other hand, when LN cells were grown in the presence of astrocytes, the small round cells were maintained at a concentration of approximately 30% of the total population. Therefore, cell-cell contact with glial cells, especially astrocytes, may be necessary to maintain the small round shape of the immature cells expressing TREM2.

Transplantation of quantum dot-labelled bone marrow-derived stem cells into the vitreous of mice with laser-induced retinal injury: Survival, integration and differentiation

Accidental laser exposure to the eyes may result in serious visual impairment due to retina degeneration. Currently limited treatment is available for laser eye injury. In the current study, we investigated the therapeutic potential of bone marrow-derived stem cells (BMSCs) for laser-induced retinal trauma. Lineage negative bone marrow cells (Lin − BMCs) were labelled with quantum dots (Qdots) to track the cells in vivo. Lin − BMCs survived well after intravitreal injection. In vivo bromodeoxyuridine (BrdU) labelling showed these cells continued to proliferate and integrate into injured retinas. Furthermore, they expressed markers that distinguished retinal pigment epithelium (RPE), endothelium, pericytes and photoreceptors. Our results suggest that BMSCs participate in the repair of retinal lesions by differentiating into retinal cells. Intravitreal transplantation of BMSCs is a potential treatment for laser-induced retinal trauma.

Reduction of inhibitor titres by infusion of FVIII gene transduced tolerogenic dendritic cells in haemophilic mice

Background: The development of anti‐factor VIII (FVIII) inhibitory antibodies is a major complication of FVIII replacement therapy in the management of patients with haemophilia A. Evidence is accumulating that tolerogenic dendritic cells (tDCs) generated in vitro can induce regulatory T cells and promote durable antigen‐specific tolerance in vivo.Objective: In this study we used human FVIII transgene expressing tDCs to reduce inhibitor formation in haemophilic mice.Methods and results: The tDCs were generated by cell sorting the CD11clowCD45RBhigh population of cells resulting from culture of lineage negative bone marrow cells in media supplemented with IL‐10 and the neural peptides VIP and PACAP38. Expression of co‐stimulatory molecules CD40, CD80 and CD86 and MHC Class II was negative or low on these cells and they remain unactivated even after stimulation with lipopolysaccharide (LPS) or transduction by foamy virus (FV) vectors. Following transduction by a FV vector expressing a B domain deleted human FVIII transgene, 3–5 × 105 tDCs were infused by tail vein injection into Bal/c haemophilic mice (tDC‐F8) weekly for two doses. Real‐time PCR showed that the transduction efficiency of our FV vector in this population of tDCs was approximately 70%. Subsequently, mice were challenged with four weekly intravenous doses of 0.2 μg rhFVIII. Mice that received no cells (Neg‐Ctrl) and mice that received an equivalent number of non‐transduced tDCs (tDC‐Ctrl) were used as controls. After immunization with rhFVIII, the spleen size, weight and total cell numbers in tDC‐F8 mice were consistently lower than in Neg‐Ctrl and tDC‐Ctrl mice by approximately 50%. Flow cytometry assessment of CD4+ splenocytes showed there were an increased number of cells expressing the regulatory T‐cell markers FOXP3, CD25, CD103, CTLA4 and GITR in tDC‐F8 treated mice. We observed a 60% and 61% reduction in the level of inhibitor titres from tDC‐F8 mice compared with Neg‐Ctrl and tDC‐Ctrl mice. Splenic CD4+ T cell proliferation in response to FVIII stimulation in cells from tDC‐F8 mice was suppressed by approximately 90% compared with proliferation of cells from Neg‐Ctrl and tDC‐Ctrl mice. We also showed that pre‐immunized mice treated with four infusions of hFVIII FV vector transduced tDCs had a reduction in their inhibitor titres by 54% (P &lt; 0.05). No significant change in inhibitor titres were seen in untreated controls or mice given four doses of untransduced tDCs.Summary: These data indicate that hFVIII gene transduced tolerogenic DCs are useful in decreasing inhibitory antibodies in haemophilic mice. More in vivo studies are in progress to confirm the antigen‐specificity and durability of these effects. Our future studies will focus on isolating and characterizing the regulatory T cell populations induced by in vivo administration of transgene modified tDCs.

Suppression of the Immune Response to Human FVIII with FVIII Transgene Modified Tolerogenic Dendritic Cells in Hemophilic Mice

The development of anti-factor VIII (FVIII) inhibitory antibodies is currently the most significant complication of FVIII replacement therapy in the management of patients with hemophilia A. Infusion of in vitro generated tolerogenic dendritic cells (tDCs) loaded with foreign antigen has been shown to promote durable antigen-specific tolerance in vivo through mechanisms that involve the induction of regulatory T cells. In this study we evaluated the ability of tDCs transduced with a human B domain deleted FVIII transgene-expressing foamy virus (FV) vector to modulate the immune response to human FVIII in both naïve and pre-immunized hemophilia A mice. The tDCs were generated by flow sorting the population of CD11clowCD45RBhigh cells produced in culture of lineage negative bone marrow cells in RPMI1640/10%FBS supplemented with IL-10 and the neural peptides VIP and PACAP38. Expression of co-stimulatory molecules CD80 and CD86 and MHC Class II was negative or low on the generated tDCs and these cells remained un-activated even after stimulation with LPS or transduction by FV vectors. These tDCs produced low levels of IL-6 and TNF-α, and high level of IL-10. Furthermore, co-culture of the vector transduced tDCs with FVIII stimulated effector T cells (Teffs) resulted in decreased proliferation of Teffs and reduced secretion of IFN-γ and IL-2. In the cultures with the transduced tDCs there was also an increase in the number of apoptotic Teffs. Naïve Balb/c hemophilia A mice were treated with 2 weekly infusions of FVIII vector transduced tDCs (tDC-F8), control tDCs (tDCs-Ctrl), or no cells (Neg-Ctrl) prior to being challenged with four weekly intravenous doses of 0.2 μg rhFVIII. Following immunization the total cellularity and weights of spleens harvested from tDC-F8 mice were consistently half that of spleens from either tDC-Ctrl or Neg-Ctrl mice. Furthermore, inhibitor titers in tDC-F8 mice were 60–61% lower than either Neg-Ctrl or tDC-Ctrl mice (p < 0.05 compared to both controls). The regulatory T cell related markers FOXP3, CD25, CD103, CTLA4 and GITR were all up-regulated on splenic CD4+ T cells from tDC-F8 mice and the CD4+ T cell proliferation response to FVIII stimulation in splenocytes from tDC-F8 mice was suppressed by approximately 90%. Moreover, the rate of apoptosis in splenic T cells from tDC-F8 mice was 33% higher than splenic T cells from either Neg-Ctrl or tDC-Ctrl mice. In pre-immunized mice, treatment with 4 weekly infusions of FVIII vector transduced tDCs lowered inhibitor titers by 54% compared to no treatment controls (p < 0.05). In contrast, treatment with untransduced tDCs had no significant effect on the inhibitor titers of pre-immunized mice. Importantly, adoptive transfer of CD4+ T cells from tDC-8 mice produced suppression of the immune response to FVIII in subsequently immunized naïve secondary recipients.. In summary, these data indicate that FVIII vector transduced tDCs are useful in suppressing the immune response to FVIII in hemophilia A mice and suggest that regulatory T cells play a role in the induced immune modulation. More in vivo studies are in progress to confirm the durability of these effects. Future studies will also focus on isolating and characterizing the regulatory T cell populations induced by in vivo administration of transgene modified tDCs.