scholarly journals Endothelial Cell-Derived Extracellular Vesicles Mitigate Radiation-Induced Hematopoietic Injury

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2581-2581
Author(s):  
Sadhna O. Piryani ◽  
Yiqun Jiao ◽  
Angel Y.F. Kam ◽  
Yang Liu ◽  
Tuan Vo-Dinh ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Nucleic Acids ◽  
Extracellular Vesicles ◽  
Cell Expansion ◽  
Lethal Dose ◽  
Flow Cytometric Analysis ◽  
Treated Group ◽  
Specific Marker ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) reside in specialized microenvironments and adjacent to endothelial cells (ECs), from which they receive regulatory cues. Following injury from radiation or chemotherapy, transplanted ECs can facilitate hematopoietic regeneration. Since these ECs do not engraft in the marrow, we hypothesize that ECs facilitate hematopoietic regeneration through secretion of extracellular vesicles (EVs). EVs are becoming recognized as a mode of cell-to-cell communication through transfer of their cargo of nucleic acids and proteins. Using ultracentrifugation, we visualized these EVs by transmission electron microscopy and measured their size to be a mean size of 160 nm (range 121-217 nm, n=18). Using flow cytometric analysis with Syto13, we demonstrate that these EVs contain nucleic acids in 30% of the particles. Quantitative real-time PCR show that these EVs express CD31 and VECadherin, and not Fibroblast Specific Marker 1 (FSP1), indicating EVs bear EC markers. At day 7 following 300 cGy, C57BL/6 cKit+Sca-1+Lineage- (KSL) cells cultured with EVs demonstrated total cell expansion and colony-forming unit cells (CFCs) compared to cultures with cytokines alone (*p< 0.0001 and 0.01, respectively, n= 3-6/group). Following myelosuppressive, sub-lethal radiation dose of 500 cGy, C57BL/6 mice treated with EVs on days 1-4 display increased marrow cellularity (*p= 0.003, n= 2-3/group), preserved vascular endothelial cells by mouse endothelial cell antigen immunohistochemistry (*p=0.03, n=2-4/group), CD150+CD48-KSL cells (*p= 0.03, n= 5/group), and increased CFCs (*p= 0.03, n= 6/group) compared to saline-treated mice. No differences were detected in these parameters when EV-treated mice were compared to mice treated with primary, cultured C57BL/6 marrow ECs. Following lethal-dose irradiation of 800 cGy, mice that received EVs on day 1-4 display prolonged survival compared to saline-treated mice (*p= 0.008 by Log-rank Analysis, n= 10/group). At day 30 following irradiation, 5 of 10 mice were alive in the EV-treated group compared to 0 of 10 in the saline-treated group. No differences in survival were observed between EV-treated and EC-treated mice (p= 0.4). Treatment of irradiated C57BL/6 hematopoietic stem/progenitor cells (HSPCs) with EVs generated from a genetically distinct strain (BALB/c mice) showed similar levels of cell expansion and CFCs compared to treatment of HSPCs from syngeneic EVs. Our findings show that syngeneic or allogenic EVs could be cell-derived therapy to deliver physiologic doses of nucleic acids and growth factors to hematopoietic cells to accelerate hematopoietic regeneration. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human adenovirus serotypes 4 and 11 show higher binding affinity and infectivity for endothelial and carcinoma cell lines than serotype 5

2003 ◽  
Vol 84 (3) ◽  
pp. 687-695 ◽  
Author(s):  
Lei-Qing Zhang ◽  
Ya-Fang Mei ◽  
Göran Wadell
Keyword(s):  
Breast Cancer ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Binding Affinity ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Human Cancer ◽  
Flow Cytometric Analysis ◽  
Carcinoma Cell Lines ◽  
Infected Cells

Adenoviruses are promising vectors for human cancer gene therapy. However, the extensively used adenoviruses serotypes 2 and 5 (Ad2 and Ad5) from species C have a major disadvantage in being highly prevalent; thus, most adults have an immunity against the two viruses. Furthermore, the expression of coxsackievirus and adenovirus receptors for Ad2 and Ad5 varies in different cells. This study aims to identify adenovirus serotypes with specific tropism for endothelial cells and epithelial tumour cells. Comparison of the binding affinities of Ad31, Ad11, Ad5, Ad37, Ad4 and Ad41, belonging to species A–F, respectively, to established cell lines of hepatoma (HepG2), breast cancer (CAMA and MG7), prostatic cancer (DU145 and LNCaP) and laryngeal cancer (Hep2), as well as to endothelial cells (HMEC), was carried out by flow cytometric analysis. Ad11 from species B showed markedly higher binding affinity than Ad5 for the endothelial cell line and all carcinoma cell lines studied. Ad4 showed a specific binding affinity for hepatoma cells and laryneal carcinoma cells. The ability of Ad11, Ad4 and Ad5 to be expressed in hepatoma, breast cancer and endothelial cell lines was studied by immunostaining and 35S-labelling of viral proteins in infected cells. Ad11 and Ad4 manifested a higher proportion of infected cells and a higher degree of hexon expression than Ad5.

Differential Role of the Transcription Factor ZBP-89 in Hemangioblast Fate Determination: ZBP-89 Is a Direct Regulator of SCL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1253-1253
Author(s):  
Xiangen Li ◽  
Carl Simon Shelley ◽  
M. Amin Arnaout
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Endothelial Cell ◽  
Embryonic Stem Cells ◽  
Flow Cytometric Analysis ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Fate Determination

Abstract Several molecular pathways have been identified that regulate distinct stages in the developmental progression from mesoderm to the formation of the hematopoietic and vascular lineages. Our previous work indicated that ectopic expression of the zinc finger transcription factor ZBP-89 promotes hematopoietic lineage development and represses endothelial cell lineage differentiation from hemangioblasts in murine embryonic stem cells. Here we evaluated the functional consequences of stable knockdown of ZBP-89 in embryonic stem cells (ESC) on hematopoietic and vascular development. Stable knock down of ZBP-89 in ESC significantly decreased the number of Blast Colony Forming Cells (BL-CFC) hemangioblasts, as well as primitive and definitive hematopoietic progenitor colonies BFU-E, GM-CFU, G-CFU, M-CFU and GEMM-CFU in vitro. In contrast, sprouting angiogenesis was markedly increased in EB cultures. Flow cytometric analysis of the lineages derived from ZBP-89 deficient EB cultures showed that the early (C-kit+Sca-1+) and definitive (CD45+) hematopoietic stem cells populations were reduced, but the endothelial cell population (CD31+ VE-Cadherin+) was increased. RT-PCR analysis of EB cultures revealed a direct correlation between the expression levels of ZBP-89 and hematopoietic markers (including SCL and Runx1) but an inverse correlation with the vascular marker CD31, with no change in Oct4 expression level. To investigate the mechanism underlying the role of ZBP-89 in hematopoiesis, the effect of ZBP-89 on expression of SCL, a master regulator of hematopoiesis, was examined. The murine SCL promoter transduced into the ZBP-89-expressing MEL cell line drove luciferase gene expression. ZBP-89 knockdown in MEL cells markedly reduced SCL expression. ChIP analysis showed that endogenous ZBP-89 protein bound directly to the murine SCL promoter in MEL cells. Thus ZBP-89 plays a central role in fate determination of hemangioblasts; its induction suppresses angiogenesis but enhances differentiation of hemangioblasts along the hematopoietic pathway, an effect mediated through the regulated expression of SCL.

IL-8 and CXCR1 Remodel the Vascular Niche to Promote Hematopoietic Stem and Progenitor Cell Engraftment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 783-783
Author(s):  
Bradley Wayne Blaser ◽  
Jessica Moore ◽  
Brian LI ◽  
Owen J. Tamplin ◽  
Vera Binder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Board Of Directors ◽  
Progenitor Cell ◽  
Transgenic Animals ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Cell Niche

Abstract The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSC/HSPC) engraftment during development and in recipients of hematopoietic stem cell transplantation (HSCT). Factors secreted by the hematopoietic microenvironment that promote HSC/HSPC engraftment in the developing zebrafish may therefore be therapeutic targets for enhancing HSC engraftment in patients undergoing HSCT. We previously described a novel behavior we called endothelial cuddling in which sinuosoidal endothelial cells of the niche make intimate interactions with stem cells. To find candidate extracellular factors regulating this behavior, gene expression profiling was performed on sorted zebrafish endothelial cells. Gene set enrichment analysis showed that expression of chemokines and TNF family members was significantly enriched in all endothelial cells. The leading edge gene sets included 16 chemokines and chemokine receptors. Thirteen of these genes were used as candidates in a gain-of-function screen to test whether overexpression was sufficient to stimulate the hematopoietic niche in favor of HSC engraftment. High level, global gene expression was induced at 36 and 48 hours post fertilization (hpf) using a heat shock-inducible system. One gene, CXCR1, enhanced HSC/HSPC engraftment when globally overexpressed (p=0.03, N=63). CXCR1 is a specific receptor for the chemokine IL-8/CXCL8 in higher vertebrates. Zebrafish IL-8 was used in similar gain of function experiments and was also sufficient to enhance HSC/HSPC engraftment (p=0.003, N=41). CXCR2 is a promiscuous chemokine receptor for IL-8, Gro-α and Gro-β and did not enhance HSC/HSPC engraftment in this system. To further characterize the effects of CXCR1 on HSC engraftment, it was overexpressed in transgenic zebrafish carrying a stem-cell specific reporter gene, Runx1:mCherry. HSC engraftment in the CHT was enhanced when CXCR1 expression was induced beginning at 36 hpf (3.0 +/- 2.0 vs 7.4 +/- 2.6 HSC per CHT) or 48 hpf (4.3 +/- 1.1 vs 9.4 +/- 3.6 HSC per CHT). Inhibition of CXCR1 signaling from 48 to 72 hpf using the selective CXCR1/2 antagonist, SB225002, decreased HSC engraftment in Runx1:mCherry animals (1.2 +/- 0.39 vs 0.4 +/- 0.2 HSC per CHT, p=0.03). We next hypothesized that overexpression of CXCR1 might also have effects on the endothelial cell niche itself. Using FLK1(VEGFR2):mCherry reporter zebrafish and 3-dimensional reconstruction of the CHT, we found that global overexpression of CXCR1 increased the volume of the endothelial cell niche (2.0 +/- 0.09 x 106 vs 2.4 +/- 0.1 x 106 μm3, p=0.005) while treatment with SB225002 reduced its volume (6.3 +/- 0.3 x 105 vs 4.9 +/- 0.5 x 105 µm3, p=0.04). Finally, we asked if CHT remodeling would still be enhanced if CXCR1 were constitutively expressed only within the endothelial cell niche. FLK1:CXCR1; FLK1:mCherry double transgenic animals had significantly increased CHT volume when compared with FLK1:mCherry single transgenic animals (1.1 +/- 0.05 x 106 vs 1.3 +/- 0.06 x 106 um3, p=0.02). These findings suggest a model whereby HSC/HSPCs actively participate in the remodeling of the endothelial niche via CXCR1/IL-8 in order to promote their own engraftment. Further, they suggest that CXCR1/IL-8 is a potential therapeutic target for enhancing HSC/HSPC engraftment in patients undergoing HSCT. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

In Vitro Hematopoietic and Endothelial Cell Development From Cells Expressing TEK Receptor in Murine Aorta-Gonad-Mesonephros Region

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1549-1556 ◽  
Author(s):  
Isao Hamaguchi ◽  
Xu-Ling Huang ◽  
Nobuyuki Takakura ◽  
Jun-ichi Tada ◽  
Yuji Yamaguchi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Platelet Endothelial Cell Adhesion ◽  
Stromal Cell Line ◽  
Macrophage Colony

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34+ and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1+ endothelial cells on the OP9 stromal layer, while TEK− cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

scholarly journals Endothelial cells provide a niche for placental hematopoietic stem/progenitor cell expansion through broad transcriptomic modification

2013 ◽  
Vol 11 (3) ◽  
pp. 1074-1090 ◽  
Author(s):  
Christophe M. Raynaud ◽  
Jason M. Butler ◽  
Najeeb M. Halabi ◽  
Faizzan S. Ahmad ◽  
Badereldeen Ahmed ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Expansion ◽  
Progenitor Cell ◽  
Hematopoietic Stem

scholarly journals Crizanlizumab Therapy Is Associated with Lower Levels of Circulating Extracellular Vesicles in Sickle Cell Disease Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 955-955
Author(s):  
Cristiane Maria de Souza ◽  
Carolina Lanaro ◽  
Irene Pereira dos Santos ◽  
Oladele Olatunya ◽  
Sara T Olalla Saad ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Board Of Directors ◽  
Extracellular Vesicles ◽  
Sickle Cell ◽  
Treated Group ◽  
Clinical Severity ◽  
Cell Disease ◽  
Advisory Committees ◽  
Activated Platelets

Abstract Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity. Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD. This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab. We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019). We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively). Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation. Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug. Disclosures Benites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

scholarly journals Cxcl8 Expands Hematopoietic Stem and Progenitor Cells and Alters Their Interaction with the Endothelial Niche

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 527-527
Author(s):  
Donn L Calkins ◽  
Jami L Shaffer ◽  
Emily M Teets ◽  
Alex M Belardo ◽  
Serine Avagyan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Time Lapse ◽  
Selective Advantage ◽  
Zebrafish Embryos ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Equity Ownership ◽  
Cell Niche

We currently have little understanding of the mechanisms by which hematopoietic stem and progenitor cells (HSPCs) gain a selective advantage in patients with clonal hematopoiesis and other myeloid neoplasms. The chemokine CXCL8 is elevated in a subset of patients with myeloid neoplasms. Our previous work in zebrafish has discovered a novel role for cxcl8 and its receptor, cxcr1, in supporting colonization of HSPCs within the sinusoidal endothelial cell niche of the embryonic zebrafish known as the caudal hematopoietic tissue (CHT). We hypothesized that mosaic overexpression of cxcl8 in a population of HSPCs during development would alter HSPC-niche interactions, selectively favor HSPCs expressing cxcl8 and lead to their expansion in adults. To test this hypothesis, we microinjected DNA constructs encoding cxcl8-2A-GFP or GFP alone under the control of the HSPC-specific Runx1+23 enhancer into zebrafish embryos at the single-cell stage. Time lapse fluorescence video microscopy and single-cell tracking was performed on HSPCs within the CHT. Overexpression of cxcl8 nearly doubled the amount of time HSPCs resided within the CHT when compared to expression of GFP alone as a control (cxcl8: 4.94 ± 0.86 h vs GFP: 2.54 ± 0.18 h, p=0.01, N=142 tracked cells). Substitution of WT cxcl8 with a mutant cxcl8 construct lacking the ELRCXC motif required for receptor binding reduced these effects (WT cxcl8: 6.6 ± 0.48 h vs ELRCXC-cxcl8: 5.3 ± 0.33 h, p=0.02, N=355 tracked cells). To observe HSPC-niche interactions, kdrl:mCherry endothelial cell reporter zebrafish were microinjected with Runx1+23:cxcl8-2A-GFP or Runx1+23:GFP DNA constructs. The percent of time individual HSPCs spent closely interacting with a single group of CHT endothelial cells (endothelial cell cuddling) was quantified over the period from 52 to 72 hours post-fertilization. Overexpression of cxcl8 by HSPCs increased HSPC-endothelial cell cuddling time by 30% (cxcl8: 87% vs GFP: 57%, p=0.001). To directly test competition between wild type and cxcl8 overexpressing HSPCs, zebrafish embryos were microinjected with a 1:1 molar ratio of Runx1+23:cxcl8-2A-mCherry and Runx1+23:clover DNA. Single cxcl8-2A-mCherry+ and clover+ competitor cells were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided longer within the CHT than competitor HSPCs when quantified over the period from 72 to 96 hours post-fertilization (cxcl8: 4.0 ± 0.20 h vs competitor: 2.5 ± 0.25 h, p=2.0 x 10-6, n=426 tracked cells). Single cell RNA-sequencing (scRNA-seq) of zebrafish embryos with mosaic expression of cxcl8 in HSPCs showed upregulation of cxcl12a in endothelial cells compared to endothelial cells from control embryos (p=5.19 x 10-3), suggesting a possible mechanism to explain the increased CHT residency time. Zebrafish with mosaic expression of Runx1+23:cxcl8 were raised to adulthood and the kidney marrow cells were analyzed by flow cytometry. Compared to clutchmate controls, Runx1+23:cxcl8 mosaic transgenics had a higher hematopoietic progenitor/precursor to lymphocyte ratio, suggesting a mild differentiation block and possible lineage skewing (cxcl8: 2.0 ± 0.15 vs control: 1.6 ± 0.10, p=0.048, N=25 animals). Taken together, these data support a model in which pre-malignant HSPC clones aberrantly express cxcl8 and acquire a selective advantage over normal clones through enhanced interactions with the endothelial cell niche. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

scholarly journals Efficacy and Safety of Allogeneic Double Negative T Cell As a Cellular Therapy for AML and Its Underlying Mechanism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1355-1355
Author(s):  
Jong Bok Lee ◽  
Weihsu Claire Chen ◽  
Elena Streck ◽  
Sohyeong Kang ◽  
Mark D. Minden ◽  
...  
Keyword(s):  
T Cell ◽  
Lethal Dose ◽  
Treated Group ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Double Negative ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in more than 70% of the treated AML patients, most relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoietic stem cell transplantation reduces the relapse rate and demonstrates the efficacy of a cell-mediated treatment for the chemotherapy-resistant disease. However, its wide application is limited by donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+ CD4-CD8- double negative T cells (DNTs) as a potential new therapy for AML patients, and to dissect its underlying mechanisms. Using a flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNTs expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Thirteen blast samples were obtained from chemotherapy refractory or relapsing AML patients and, of those, 9 were susceptible to DNTs. Further, the average level of specific killing mediated by DNTs against chemotherapy-susceptible and -resistant blasts were comparable (19.30% ± 3.34% and 15.91% ± 3.63%, respectively). The anti-leukemia activity of DNTs was further validated in AML-NSG xenograft models. A single infusion of DNTs into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. While a single injection of DNTs resulted in a significant reduction of leukemia burden from 30% to 12.8%, it was further reduced to 2.6% after three injections of DNTs. Furthermore, the survival of NSG mice administered with a lethal dose of AML cell line, MV4-11, in DNT-treated group was significantly superior to the untreated group (57% vs. 0% survival on day 34, respectively). Although residual blasts were observed from the DNT -treated group, their susceptibility to DNT cell-mediated cytotoxicity remained comparable to blasts obtained from the PBS-treated group and primary AML blasts initially used for engraftment, suggesting that AMLs do not develop resistance to DNT-mediated cytotoxicity. In contrast to its potent cytolytic activity against leukemic cells, DNTs did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. Administration of allogeneic DNTs into NSG mice engrafted with human HSPC had no effect on the engraftment level of human hematopoietic cells and their differentiation into different lineages. Further, in contrast to human PBMC, infusion of human DNTs did not cause xenogeneic GvHD in mice, collectively demonstrating the selective cytotoxic activity of allogeneic DNTs against leukemic cells. Using blocking assays, we showed that HLA-class I, NKG2D, and DNAM-1 were involved in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor did not play a significant role. We detected high levels of IFNγ release by DNTs upon encounter of susceptible AML targets. While IFNγ treatment alone did not induce AML cell death, neutralizing IFNγ reduced and pretreating AML cells with recombinant IFNγ increased their susceptibility to DNT cell killing. IFNγ treatment induced higher expressions of NKG2D and DNAM-1 ligands and blocking of NKG2D and DNAM-1 partially abrogated the effect of IFNγ on the AML-DNT interaction. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed the important molecules for the anti-leukemic activity of DNTs. Disclosures No relevant conflicts of interest to declare.

Myeloid Lineage-Restricted Progenitors Contribute to Vascular Endothelium.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2267-2267
Author(s):  
Alexis S. Bailey ◽  
Holger Willenbring ◽  
Shuguang Jiang ◽  
David A. Schroeder ◽  
Daniel A. Anderson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Fusion ◽  
Reporter Gene ◽  
Radiation Injury ◽  
Cre Recombinase ◽  
Reporter Gene Expression ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

Abstract Endothelial progenitors of hematopoietic origin have a role in promoting recovery from vascular injury. Recently we have shown that following radiation damage, transplanted hematopoietic stem cells (HSCs) give rise to endothelial cell outcomes at the clonal level. This raises the question of whether prospectively identifiable HSC progeny have endothelial cell activity. Using β-galactosidase and EGFP expression as donor markers, we show that lineage-restricted myeloid progenitors can give rise to endothelial cells. Transplanted common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) gave rise to similar numbers of endothelial cells in liver portal vein branches. Donor-derived endothelial cells expressing functional endothelial markers including CD31, von Willebrand factor (vWF) and Tie-2 were clearly distinguished from mature hematopoietic cells by the absence of CD45 and F4/80 expression. Confocal microscopy showed the co-localization of donor and endothelial cell marker expression in individual cells so that endothelial cells could be discriminated from perivascular cells. To evaluate cell fusion as a potential mechanism for the emergence of endothelial cells of hematopoietic origin, HSCs transgenic for EGFP and Tie-2-Cre recombinase were transplanted into ROSA26 reporter (R26R) mice. Activation of β-galactosidase reporter gene expression, indicative of cell fusion with a donor-derived hematopoietic cell, could be detected in hepatocytes but not in endothelial cells. However, due to the tissue-specificity of the Tie-2 promoter, endothelial fusion products in which the donor nucleus had failed to activate the Tie-2-driven Cre recombinase would have also lacked detectable reporter gene expression. To ensure that the absence of β-galactosidase expression in the donor-derived endothelial cells was not due to insufficient nuclear reprogramming, HSCs transgenic for EGFP and R26R were transplanted into Tie-2-Cre recipients. Donor-derived endothelial cells were again negative for both β-galactosidase expression and activity. To determine whether radiation injury is required for the incorporation of donor cells into the endothelium, a parabiosis model was utilized. Both long-term donor-derived endothelial cell outcomes and multilineage hematopoiesis were detected in parabiotic mice. These results demonstrate that CMPs and further lineage-restricted GMPs are capable of differentiating into endothelial cells in the absence of cell fusion. Importantly, radiation injury is not required for endothelial cell engraftment, indicating that circulating progenitors contribute to angiogenesis during steady-state conditions.

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