Heterogeneity in the Making of Blood

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-27-SCI-27
Author(s):  
David T. Scadden
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Niche ◽  
Cell Selection ◽  
Hematopoietic Progenitor ◽  
Marrow Space ◽  
And Function ◽  
Osteolineage Cells

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell Intrinsic and Extrinsic Mechanisms Mediate the Role of c-Myc Haploinsufficiency in Controlling HSC Fate

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2561-2561
Author(s):  
Yue Sheng ◽  
Chunjie Yu ◽  
Rui Ma ◽  
Zhijian Qian
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
White Blood Count ◽  
Bone Marrow Niche ◽  
Severe Anemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract It has been shown that loss of c-Myc leads to accumulation of hematopoietic stem cells (HSCs) and severe cytopenia as a consequence of a blockage of HSC differentiation. Here we report a role of c-Myc haploinsufficiency in regulating HSC quiescence and self-renewal. We showed that c-Myc haploinsufficient mice displayed decreased white blood count and number of lymphocytes with normal myeloid cell differentiation. The number of HSCs and hematopoietic progenitor cells (HPCs) were all decreased significantly in c-Myc haploinsufficient mice as compared with control mice. We found that c-Myc haploinsufficiency inhibited HSC self-renewal capacity, increased proliferation and decreased quiescence of HSCs in vivo. By transplantation assays, we showed that c-Myc haploinsufficiency has extrinsic and intrinsic effects on the maintenance of HSCs in vivo. Our study suggests that loss of c-Myc activity and reduced dosage of c-Myc have distinct effects on HSC functions. c-Myc is a critical downstream mediator of the Wnt/b-catenin pathway. We showed that c-Myc haploinsufficiency is sufficient to prevent severe anemia in Apc heterozygous mice, and to significantly prolong the survival of Apc heterozygous mice. In addition, treatment of Apc haploinsufficient mice by a c-Myc inhibitor significantly reversed anemia in Apc-deficient mice. By transplantation assay, we further demonstrated that reduced expression of c-Myc in the bone marrow niche is responsible for prevention of severe anemia in Apc-deficient mice. However, we found that reduction of c-Myc by loss of a single allele of c-Myc did not rescue defective self-renewal capacity of Apc haploinsufficient HSCs. Taken together, our studies indicate that c-Myc mediates the function of the Wnt/b-catenin signaling pathway in bone marrow niche but not in HSCs. Disclosures No relevant conflicts of interest to declare.

CD44v6 Is Required For In Vivo Tumorigenesis Of Human AML and MM Cells: Role Of Microenvironmental Signals and Therapeutic Implications

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 605-605 ◽  
Author(s):  
Benedetta Nicolis di Robilant ◽  
Monica Casucci ◽  
Laura Falcone ◽  
Barbara Camisa ◽  
Bernhard Gentner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Conflicts Of Interest ◽  
Human Leukemia ◽  
Vegf Receptor ◽  
Durable Response ◽  
Wide Range ◽  
Cd44v6 Expression

Abstract Background Targeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. Aim To preclinically validate CD44v6 as a therapeutic target in AML and MM Results By FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P<0.001). These results were confirmed by using THP-1 cells (P<0.001) and primary leukemic blasts (P<0.01). Hypothesizing that CD44v6 may be crucial for in vivo tumorigenesis by cooperating with RTKs, we set-up a co-culture system with BM-derived mesenchymal stromal cells (MSCs), which are producers of VEGF and the c-Met ligand HGF. MSCs protected a wide range of tumor cells, including primary leukemic blasts, from spontaneous apoptosis (P<0.05) and from apoptosis induced by Ara-C or daunorubicin (P<0.01), or bortezomib in the case of MM cells (P<0.001). Comparable results were obtained by using MSC supernatants, hinting to a causative soluble factor, which was neither VEGF nor HGF, as demonstrated by inhibition experiments with bevacizumab and crizotinib, respectively. Noteworthy, MSCs or their supernatants prompted a significant up-regulation of CD44v6 expression levels (P<0.01). Most importantly, preventing CD44v6 up-regulation on tumor cells by shRNA silencing restored their sensitivity to spontaneous and drug-induced apoptosis (P<0.01). Conclusions These results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1339-1339
Author(s):  
Namit Sharma ◽  
Pan Zhongda ◽  
Tracy Lauren Smith ◽  
Savar Kaul ◽  
Emilie Ernoult ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Apoptotic Cells ◽  
Direct Role

Abstract Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase. Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. Disclosures No relevant conflicts of interest to declare.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Modulation in the Expression of p70S6 Kinase Impairs the Engraftment and Self-Renewal of Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 252-252
Author(s):  
Joydeep Ghosh ◽  
Baskar Ramdas ◽  
Anindya Chatterjee ◽  
Peilin Ma ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Mtorc1 Pathway ◽  
And Function

Abstract Regulation of hematopoietic stem cell (HSC) function(s) via the mammalian target of rapamycin complex1 (mTORC1) and its upstream regulators including PI3K and Akt has been described before. To this end, we and others have shown that hyperactivation and deficiency of the PI3K-mTORC1 pathway results in altered development, maintenance and function(s) of HSCs. However, the role of downstream effector of mTORC1, p70S6 kinase (S6K1), in HSC development and functions is unknown. Previous studies have implicated S6K1 as a regulator of ageing, by virtue of its ability to regulate cellular metabolic processes as well as protein translation. In certain cells, however S6K1 regulates cell survival and also acts as a negative regulator of PI3K-mTORC1 pathway, thus creating a negative feedback loop. Thus, how S6K1 impacts HSC ageing and stem cell functions remains an enigma. We have assessed the role of S6K1 in HSC development and function under steady-state as well as during recovery of hematopoietic system following myelosuppressive stress. We used a genetic model of S6K1 knockout mice (S6K1-/-). S6K1 deficiency in bone marrow hematopoietic cells resulted in decrease of absolute number of bone marrow hematopoietic progenitor cells as well as HSCs (Lin- Sca1+ c-Kit+; LSK) were significantly reduced relative to controls (n=14 in each group, p<0.04). Interestingly, in vitro, hematopoietic progenitor cells from S6K1-/- mice showed increased colony forming ability in response to cytokines which was associated with hyperactivation of Akt and ERK MAP kinase. To determine whether the reduced number of HSCs in S6K1-/- mice was due to deficiency of S6K1 in bone marrow microenvironment, we transplanted WT hematopoietic bone marrow cells into lethally irradiated WT or S6K1-/- mice. S6K1-/- mice transplanted with WT hematopoietic cells showed similar bone marrow cellularity and HSC numbers compared to controls suggesting that the bone marrow hypocellularity and reduced HSCs numbers in S6K1-/- mice were due to a cell intrinsic defect. To assess whether the reduced HSC number in S6K1-/- mice impacted the recovery of hematopoietic system following stress, WT and S6K1-/- mice were treated with a single dose of 5-fluorouracil (5-FU). In response to myelosuppressive stress, S6K1 deficiency resulted in increased frequency of HSCs in bone marrow despite a significant reduction in overall cellularity (n=12 in each group, p<0.02). Following administration of 5-FU, S6K1 deficiency resulted in increased cell cycle progression of HSCs in bone marrow and showed increased expression of CDK4 and CDK6 as compared to control suggesting that 5-FU administration results in upregulation of cell cycle regulatory genes in S6K1 deficient HSCs. Moreover, S6K1-/- mice showed more sensitivity to repeated injections of 5-FU (n=11 WT, 15 S6K1-/-, p<0.01). Given the differential role of S6K1 in HSCs and mature progenitors, we assessed the effect of S6K1 deficiency in HSC function. We performed competitive repopulation assay using S6K1 deficient HSCs. When transplanted into lethally irradiated primary and secondary recipients, S6K1 deficient HSCs show significantly reduced engraftment relative to controls (n=11-13 in each group; p<0.05). Interestingly, overexpression of S6K1 in wild type HSCs also resulted in reduced engraftment of HSCs in primary and secondary transplant recipients, suggesting that S6K1 overexpression in HSCs leads to decreased self-renewal. In summary, our study identifies S6K1 as a critical regulator of hematopoietic stem cell development and functions both under steady-state conditions as well as under conditions of genotoxic stress. Using both gain of function and loss of function approaches, we demonstrate that the level of expression and activation of S6K1in HSCs plays a critical role in the maintenance of HSC self-renewal and engraftment. Disclosures No relevant conflicts of interest to declare.

Novel Pleiotropic Effects of Thrombin in Regulation of Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells – Identification of Negative PAR1 and PAR3 Receptor Crosstalk.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 336-336
Author(s):  
Marcin Wysoczynski ◽  
Rui Liu ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Metastatic Potential ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Coagulation Cascade ◽  
Receptor Crosstalk

Abstract Abstract 336 Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM) to this degrees that it may mimic acute lymphocytic leukemia. We identified chemokines and growth factors (e.g., SDF-1, HGF) that play an important role in RMS metastasis (Blood 2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res. 2007;67:2131). Novel evidence however accumulates that metastatic process for many tumors may be modulated by the components of coagulation cascade (CC) (e.g., thrombin, activated platelets). Thus, we become interested on a role of CC in modulating metastasis of RMS cells. First, we learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generation of thrombin. Thrombin activated in tumor microenvironment activates platelets that release microvesicles. We observed that platelet derived microvesicles (PMV) transfer to RMS cells several platelet integrin receptors (e.g., α2β3) important for RMS cell interaction with endothelium, and thus increase their adhesive potential to endothelial cells. To support this, we noticed that RMS cells covered with PMV showed higher metastatic potential after intravenous injection into immunodeficient SCID mice. We also found that PMV also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Next we learned that all 10 human RMS cell lines investigated in our studies express functional PAR1 and PAR3 receptors. To support this, we observed in thrombin stimulated RMS cells phosphorylation of MAPKp42/44 and MAPKp38. To our surprise however, in in vitro experiments thrombin decreased RMS chemotactic response to conditioned media from bone marrow fibroblast and PMVs. Furthermore, we didn't observe any effect of thrombin on proliferation, survival and expression of pro-angiogenic factors in RMS cells. Thrombin also decreased adhesion of RMS cells to fibronectin and bone marrow stroma cells. In contrast PAR1 specific agonist TRAP-6 stimulated proliferation of RMS cells. Different responsiveness to thrombin and TRAP-6 stimulation could be explained by negative modulatory role of PAR3 receptor in response to stimulation by thrombin. Thus, to learn more on a role of PAR1 and PAR3 in RMS proliferation/metastasis we knock-down both receptors by employing shRNA strategy. We observed that PAR1-/- receptor RMS cells that express intact PAR3 cells formed in vivo smaller tumors as compared to unmodified control cells. On the other hand, PAR3-/- RMS cells that express functional PAR1 began to proliferate robust in response to thrombin. In conclusion, we demonstrate that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis. Finally, our data also supports a negative regulatory role of thrombin-PAR3 axis in proliferation of RMS. Disclosures: No relevant conflicts of interest to declare.

Distinct Role of Antigen-Specific Tc1 and Tc17 Cells in Tumor Eradication In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1910-1910
Author(s):  
Yu Yu ◽  
Hyun Il Cho ◽  
Dapeng Wang ◽  
Kane Kaosaard ◽  
Claudio Anasetti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Conflicts Of Interest ◽  
B16 Melanoma ◽  
Host Cells ◽  
Tumor Bearing ◽  
Tc17 Cells

Abstract Abstract 1910 Background: Adoptive cell transfer (ACT) of tumor-reactive T cells is one of the most promising approaches for the treatment of established melanoma. Recently, limited studies provide some evidence that Th/Tc17 cells may also have potent anti-tumor activities, but the conclusion is far from reach. Methods: Human gp10025-specific Tc1 or Tc17 cells were generated from pmel-1 transgeneic mice and used as cell source for ACT. Luciferase-transduced B16 melanoma was intravenously injected into C57BL/6 mice to establish lung-metastasis. After 7 days, tumor-bearing mice were lethally irradiated and transferred gp-10025 specific Tc1 or Tc17 cells in the combination of syngeneic bone marrow. Survival of those tumor-bearing mice was monitored daily, and tumor growth was monitored weekly using in vivo bioluminescent imaging (BLI). Donor T-cell expansion and cytokine secretion from the spleen and lung of tumor bearing mice were analyzed using flow cytometry and ELISPOT assays. To evaluate the role of IFNγ in anti-tumor immunity, we used a B16 melanoma cell line that was transduced with a plasmid encoding a dominant-negative IFNγ receptor (B16-IFNγRDN), and IFNγR knockout mice as tumor-bearers. Results: As expected, irradiation and transfer of syngeneic bone marrow had little or no effect on established melanoma. Adoptive transfer of tumor-specific Tc17 cells significantly suppressed the tumor growth, whereas Tc1 cells induced long-term regression of established melanoma. After ACT, Tc1 cells maintained their phenotype to produce IFNγ. However, Tc17 cells largely preserved their ability to produce IL-17, but a subset of them secreted IFNγ, indicating the plasticity of Tc17 cells in vivo. Mechanistically, Tc1 cells executed their anti-tumor immunity primarily through the direct effect of IFNγ on melanoma cells because Tc1 cells had essentially no effect on B16-IFNγRDN tumor. However, Tc1 cells had a similar therapeutic effect on IFNγR knockout as wild type mice, indicating that IFNγ signaling in host cells was not critical. In contrast, despite the fact that Tc17 cells also secreted IFNγ, Tc17-mediated anti-tumor immunity was independent of the effect through IFNγ. Ironically, IFNγ was inhibitory to Tc17-mediated anti-tumor activity. Conclusions: Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective anti-tumor immunity, but Tc1 is superior to Tc17. These findings also demonstrated for the distinct effect mechanisms of antigen-specific Tc1 and Tc17 cells in anti-tumor response, and direct IFNγ signaling on tumor cells is a key effect to eradicate established tumors mediated by Tc1 cells. Disclosures: No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cathepsin B Mediated Lysosomal Degradation in Macrophages Controls the Pharmacokinetics of the Therapeutic Protein Asparaginase

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3768-3768
Author(s):  
Laurens T Van Der Meer ◽  
Samantha YA Terry ◽  
Dorette van Ingen Schenau ◽  
Kiki Andree ◽  
Gerben M Franssen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Vivo Imaging ◽  
Cathepsin B ◽  
Rational Design ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Protein ◽  
Bone Marrow Niche ◽  
Lysosomal Protease

Abstract Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Murine Fetal Bone Marrow HSPCs Undergo a Dramatic Shift in Frequency at Birth

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2471-2471
Author(s):  
Trent Hall ◽  
Pramika Sriram ◽  
Shannon McKinney-Freeman
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Absolute Number ◽  
Global Changes ◽  
Bone Marrow Niche ◽  
Mouse Development ◽  
Hematopoietic Stem ◽  
Fetal Bone ◽  
And Function

Hematopoietic stem cells (HSCs) give rise to all cells of the hematopoietic system and are classically defined by their ability to stably engraft and reconstitute the blood system of ablated recipients after transplantation. The first transplantable HSCs arise from hemogenic endothelium at embryonic day 10.5 (E10.5) of mouse development and migrate to the fetal liver (FL), where they undergo a robust expansion followed by a second migration to the fetal bone marrow (FBM) at E15.5. The dynamics of hematopoiesis within the FBM has been largely unexplored. To gain a better understanding of FBM hematopoiesis, we catalogued the frequency, absolute numbers, phenotype and function of HSPCs in murine FBM from E15.5 through post-natal day 28 (P28). To avoid assumptions regarding HSPC location during fetal and neonatal development, we pooled bone marrow from the entire fetal skeleton for these studies. HSCs were rare in the FBM, ranging from 70-150 total HSCs at E15.5-E17.5, followed by a burst at E18.5 to 2,200 total HSCs. The frequency and absolute number of HSCs in the bone marrow steadily increased from E18.5 to P6, followed by a continual increase in the absolute number of HSCs from P6 to adulthood. This may reflect the dynamics of HSC cycling, or an influx or expansion of more differentiated progenitors in the fetal and neonatal bone marrow. We also found that the most prevalent hematopoietic stem and progenitor cell (HSPC) population within Lineage-Sca-1+c-Kit+ (LSK) cells in E15.5-E18.5 FBM was Flt3-CD48+CD150+ cells (MPP2). MPP2 cells, which are a megakaryocyte-biased multipotent progenitor population, comprised up to 75% of the LSK compartment at these time points, compared to 3% in adult bone marrow. The percentage of MPP2 cells dropped abruptly and dramatically right before birth (e.g. to 17% at E19) and continued to drop until adulthood. Transplantation of limiting numbers of MPP2 cells revealed that E16.5, E18.5, and P0 MPP2s displayed no repopulating potential, while adult MPP2s showed transient reconstitution of irradiated recipients. Fetal bone marrow MPPs displayed no colony potential in single-cell methylcellulose colony assays until E18.5 and P0, with a bias for erythroid-megakaryocyte colonies. Therefore, fetal bone marrow MPP2s are functionally distinct from their adult counterparts. Whole fetal bone marrow transplants showed that the first transplantable FBM HSCs appeared at E16.5, with up to 75% reconstitution in the peripheral blood (PB) of irradiated recipients. E16.5 FBM HSCs also displayed secondary transplantation activity, while E15.5 FBM HSCs displayed limited ability to reconstitute the PB of primary recipients. In sum, our studies reveal that until birth the predominant HSPC population in the fetal bone marrow is an immunophenotypic MPP2 that is functionally distinct from adult MPP2s, and that HSCs do not begin to accumulate in significant numbers until right before birth. These studies suggest the presence of key mechanisms during birth that influence the HSPC landscape of the fetal and neonatal bone marrow, and work is currently underway to systematically characterize global changes in the bone marrow niche during parturition. Disclosures No relevant conflicts of interest to declare.

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