689 AAV5 Mediated CD39/CD73 Expression Effects of Adenosine and Regulation of Its Receptors in Primary Cells From Healthy Donors and Rheumatoid Arthritis Patients Molecular Therapy Volume 22, Suppleme[.]
HEMATOLOGIC & IMMUNOLOGIC DISEASES II growth of erythroid colonies at low erythropoietin concentration Erythropoietin dependence was maintained since colonies did not grow in the presence of anti-erythropoietin blocking antibodies The assessments of the efficacy and the safety of the new LentiGlobin BB305-tEpoR vector in mice and cynomolgus monkey transplant models after reduced conditioning are ongoing 688 Functional Niche Competition Between Normal and Leukemic Stem Cells Chen Glait-Santar,1 Xingmin Feng,1 Jichun Chen,1 Ronan Desmond,1 Benjamin Mizukawa,2 James C Mulloy,2 Neal S Young,1 Cynthia E Dunbar.1 Hematology Branch, National Heart, Lung, and Blood Institute,National Institutes of Health, Bethesda, MD; 2Cancer and Blood Disease Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH Hematopoietic stem cells (HSCs) reside in a specialized niche that regulates their proliferative capacity and their fate The functions of HSCs are regulated by both intrinsic signals from the cells themselves and from surrounding bone microenvironment These signals enable tight regulation of stem cell pool size together with steady-state and injury-responsive hematopoiesis There is increasing evidence for similar roles of marrow niches in impacting on the behavior of leukemic stem cells, for instance protecting LSCs from cytotoxic drugs, however whether normal HSCs and leukemic cells reside in or could compete for the same marrow niche is unknown We used a murine competitive repopulation model and MLL-AF9 myeloid leukemia to investigate whether normal and leukemic stem cells compete for the same niches Mice were transplanted with progenitor cells transduced with an MLL-AF9 vector expressing GFP in a murine syngeneic model We co-transplanted a fixed number of MLL-AF9 cells generated by transduction of primary murine progenitors with MLL-AF9/GFP vector along with increasing numbers of normal whole bone marrow (WBM) cells, derived from syngeneic or dsRed transgenic mice, into irradiated recipient mice WBM cell doses were above the threshold permitting full hematopoietic recovery from irradiation Control groups received MLL-AF9 and normal WBM cells only Time to a moribund state, spleen weight and marrow cellularity at sacrifice as well as leukemic infiltration were assessed Interestingly, survival was increased and leukemic progression delayed proportional to increasing doses of normal WBM cells, in three independent experiments Confocal microscopy confirmed co-localization of engrafting WBM and MLL-AF9 progenitors in proximity with marrow microenvironment MLL-AF9 cells isolated from transplanted recipient marrows were cycling more actively in the presence of increasing doses of WBM, suggesting competitive exclusion from the protective marrow niche Osteopontin (OPN) is a cytokine produced S266 by niche components and demonstrated to promote anchoring and quiescence of normal and acute lymphoblastic leukemia stem cells Quantification of OPN expression in BM supernatants collected from femurs pointed to higher OPN levels correlated with lower cycling of recovered ALL-MF9 cells measured at the time mice were moribund The competitive model suggests that normal and myeloid leukemic stem cells compete for the same functional niche, thus niche manipulation could impact on response to anti-leukemic therapies, and the extent of normal hematopoiesis could impact on outcomes, informing approaches to cell dose in the context of stem cell transplantation 689 AAV5-Mediated CD39/CD73 Expression: Effects of Adenosine and Regulation of Its Receptors in Primary Cells From Healthy Donors and Rheumatoid Arthritis Patients Susanne A Snoek,1,2 Ciska Braam,1,2 Ronne Brunekreef,1,2 Lisa van Baarsen,2 Niels Broekstra,1,2 Jan van Ittersum,1,2 Paul P Tak,1,2 Margriet J Vervoordeldonk,1,2 Jonathan D Finn.1,2 Arthrogen B.V., Amsterdam, Netherlands; 2Academic Medical Center, Amsterdam, Netherlands The conversion of extracellular ATP to adenosine is an important mechanism of immune suppression by Tregs, and this is done by expression of ENTPD1 (CD39) and 5NTE1 (CD73) CD39 is a membrane bound ATPase that converts extracellular ATP and ADP to AMP, whereas CD73 is a membrane bound ecto-nucleotidase that converts AMP to adenosine We have found evidence suggesting that the balance between pro-inflammatory ATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis (RA) patients Therefore, we aim to ameliorate this balance by AAV mediated expression of CD39 and CD73 Here we present data investigating the effects of adenosine and expression of its receptors (ADORA-1, -2A, -2B, -3) in two of the major cell types involved in the pathogenesis of RA, monocytes and fibroblast-like synoviocytes (FLS) Also, we show that AAV-mediated expression of CD39 and CD73 is efficacious in primary cell based in vitro inflammation assays Primary monocytes were isolated from healthy donor blood and FLS from synovial biopsies from RA patients These cell types showed a distinct ADORA expression profile, with ADORA1/2B predominant on FLS, and ADORA3/2B predominant on monocytes Interestingly, both cell types showed a striking upregulation of ADORA2A, but not of ADORA-1, -2B or -3, following stimulation with LPS (monocytes: 102-fold) or TNF/IL-1β (FLS: 432-fold) When monocytes were stimulated with LPS, adenosine significantly reduced pro-inflammatory cytokine production, resulting in a >70% decrease in CCL2 and TNF In similar studies using FLS, adenosine was found to have a much more modest effect, showing only a 2-fold over SCGM over weeks Cytokine concentrations and timing were optimized to maximize expansion and differentiation ability All cells were expanded in KIT ligand, Flt3 ligand, GM-CSF, and IL-6 (K/F/GM/6) In protocol 1, M-CSF (M) was added after weeks of expansion In protocol 2, cells were moved after one week of expansion into M-CSF and IL-1b (M/1b) containing media for differentiation Differentiation was identified by flow cytometry for CD14 and CD11b Differentiation was consistently higher at two weeks in M/1b media compared to K/F/ GM/6/M media Cell morphology evaluated on cytospins confirmed enhanced differentiation in the M/1b medium Four BM samples of ~ 20ml were also obtained from hPAP patients with CSF2RA mutations and CD34+ cells were isolated A mean of 10x106 CD34+ cells were isolated with a >95% CD34+ purity and viability CD34+ cells were transduced with CSF2RA-GFP SIN lentiviral vector at 1x10^7 iu/ml with 25% GFP transduction efficiency Transduced cells had increased expansion compared to mock with 16.6 fold and 3.3-fold expansion, respectively Expanded transduced cells were able to differentiate as evidenced by expression of CD11b (68%) and CD14 (63%) The cells had an increased phagocytosis of E coli beads (44-65%) compared to control cells (22%) and restored GM-CSF signaling through Stat5 Together, these data demonstrate the feasibility of expanding CD34+ cells from normal or gene corrected hPAP patients, and highlight the potential of this approach as a treatment for hPAP Future studies will evaluate the in vivo potential of these cells to functionally correct hPAP in an immune deficient mouse model of hPAP S267 ... expression of CD39 and CD73 from FLS is effective in reducing pro-inflammatory cytokine production in a THP1 based in vitro inflammation assay Thus, the modulation of ATP :adenosine levels by CD39 and. .. 5.3% in hypoxia and 16.8 ± 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 ± 6.7% in hypoxia and 8.4 ± 2.3% in normoxia, p=0.47) When considering the bulk of CD34+ cells, ... GFP vectors in hypoxia and TdTomato vectors in normoxia These conditions were reversed in one animal Cells from both fractions were combined and injected intravenously in lethally irradiated