Potent Strategy for Treatmet of Gvhd and Autoimmune-Diseased by Infusion of Donor-Typed or Even Third-Party Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment

Abstract Abstract 2777 We have reported that bone marrow derived dendritic cells with psoralen and UVA (PUVA-DCs) treatment acquired tolerogenicity in mice. With the purpose of potential application of PUVA-DCs in a clinical hematopoietic stem cell transplantations (HSCT) for graft-versus-host disease (GVHD), we showed that mixed lymphocyte reaction (MLR) was strongly inhibited when PUVA-DCs from the stimulator strain were added to the coculture (Stimulator (S): conventional DCs obtained from C57BL/6, Responder (R): splenocytes obtained from Balb/c, PUVA-DCs: C57BL/6). This suggests that infusion of host-typed PUVA-DCs would become a novel therapeutic approach for GVHD. However utilizing host-typed DCs has problems because of leukemic cell contaminations or low efficiency of cell culture from the patients receiving repetitive chemotherapy. Therefore next concern is whether PUVA-DCs generated from BM donor or even strangers would have same tolerogenicity as host-typed PUVA-DCs do. To test this, we performed MLR by adding PUVA-DC generated from the same strain of responder or third party strain (S: conventional DCs obtained from C57BL/6, R: splenocytes obtained from Balb/c, PUVA-DC: C57BL/6 or C3H). Proliferation was significantly inhibited when PUVA-DC generated from the stimulator strain were added to the coculture (p<0.05). Also significant inhibition was observed (p<0.05) when adding PUVA-DCs generated from third party, suggesting that PUVA-DCs have tolerogenicity in a MHC-independent manner. To clarify the mechanisms of how PUVA-DCs induce tolerogenicity, we performed MLR as mentioned above with the addition of neutralizing antibodies against IL-10 or TGF-beta1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner. This is the first report that PUVA-DCs prepared not only from host-typed but from donor-typed or even third-party could induce strong inhibition of alloreaction. Tolerogenic DCs prepared previously by several ways could not induce inhibition of alloreaction in vitro when these cells were prepared from donor-typed or third-party strains in mice. To apply tolerogenic dendritic cells for GVHD in clinical settings, it is necessary to obtain sufficient doses of PUVA-DCs with ease and safety guaranteed. Therefore in the future PUVA-DCs generated even from HLA mismatched iPS cells would be a promising approach. In conclusion, infusion of PUVA-DCs from donor-typed or even third party strain could have a potent strategy for treatment of lethal GVHD and autoimmune diseases. Disclosure: No relevant conflicts of interest to declare.

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Extracellular DEK Is a Novel Chemotactic Agent of Lineage Negative, Sca-1 Positive, c-Kit Positive Bone Marrow Cells

Blood ◽

10.1182/blood.v124.21.1583.1583 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1583-1583

Author(s):

Maegan L. Capitano ◽

Nirit Mor-Vaknin ◽

Maureen Legendre ◽

David Markovitz ◽

Hal E. Broxmeyer

Keyword(s):

Bone Marrow ◽

G Protein ◽

Pertussis Toxin ◽

Neutralizing Antibodies ◽

Conflicts Of Interest ◽

Regulation Of Transcription ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Hematopoietic Stem ◽

G Protein Coupled

Abstract DEK, a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is known to be secreted by macrophages and act as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK is known to function as a chemotactic factor that attracts neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells, SDF-1 being the most potent of proteins with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative, sca-1 positive, c-kit positive (LSK) mouse bone marrow and neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced migration of LSK cells at a dose of 100ng/mL, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ cells. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with the maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration was observed when IL-8 or MIP-2 was utilized. When Ly6G+ cells were used CXCR2 neutralizing antibodies blocked the migration of Ly6G+ cells towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ migration towards SDF-1. CXCR2 is known to be a G protein-coupled receptor and this interaction can be blocked through the use of pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro. Thus, DEK may be involved in migration and homing of HSCs and HPCs. Disclosures No relevant conflicts of interest to declare.

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Strong Inhibition of Alloreaction by Newly Generated Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment.

Blood ◽

10.1182/blood.v114.22.3686.3686 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3686-3686

Author(s):

Hideaki Maeba ◽

Ryosei Nishimura ◽

Rie Kuroda ◽

Raita Araki ◽

Shintaro Mase ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Adaptive Immune Response ◽

Cell Contact ◽

Inhibition Of Alloreaction

Abstract Abstract 3686 Poster Board III-622 Dendritic cells (DCs) are a heterogenous population of antigen-presenting cells (APCs) that contribute to innate immunity and initiate the adaptive immune response. In addition, recent studies demonstrated the existence of tolerogenic DCs (TDCs) that suppress the immunoreaction. Although the tolerogenic mechanisms are not fully understood, there are some methods reported to generate TDCs from conventional DCs by using cytokines such as IL-10/TGF-beta, vasoactive intestinal peptide (VIP), and IL-21. With the purpose of potential application of TDCs in a clinical bone marrow transplantation for preventing graft-versus-host disease (GVHD), which is caused by strong immunoreaction between host-typed conventional DCs and donor-typed naïve T cells, the establishment of much safer and easier, and more efficient TDCs culture system would be needed. To this end, we investigated whether treatment of conventional DCs with psoralen plus UVA (PUVA), which is widely and safely available in the treatment of some human immune disease and organ transplantation for preventing graft rejection, induced a subset of highly potent TDCs. Bone marrow cells obtained from Balb/c (H-2d) or C57BL/6 (H-2b) were incubated in complete RPMI containing GM-CSF for 10 days to generate bone marrow derived DCs (BM-DCs). BM-DCs were cultured with Psoralen (200 ng/mL) for 30 miniutes and then exposed to UVA light (2J/cm2). After 24 hours UVA irradiation, PUVA-treated DCs were collected and used in all experiments. First, PUVA-treated or untreated DCs with irradiatin were used as stimulator for allogenic splenocytes in mixed leukocyte reactions (MLR). The immunostimulatory capacity of PUVA-treated DCs was significantly diminished compared to those of untreated DCs (p<0.01). The expression levels of CD80 and CD86 by FACS, both of which are costimulatory molecules for T cell activation, was significantly reduced after PUVA treatment (p<0.05). This might explain for the induction of hyporesponsiveness in part. Next we further evaluated whether PUVA-treated DCs directly suppress T cell alloreaciton by cell-to-cell contact. Proliferation was inhibited when PUVA-treated DCs from the stimulator strain were added to the coculture, with a maximum reduction in proliferation of 95% at a 1:1 or higher ratio of untreated DCs to PUVA-treated DCs (Attached file). In conclusion, PUVA-treated DCs directly inhibit T cell alloreaction. Infusion of host-typed PUVA-treated DCs would be potent strategy for preventing lethal acute GVHD. Disclosures: No relevant conflicts of interest to declare.

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Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood.v128.22.2641.2641 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2641-2641

Author(s):

Lorena Lobo Figueiredo-Pontes ◽

Robert S. Welner ◽

Miroslava Kardosova ◽

Hong Zhang ◽

Meritxell Alberich-Jorda ◽

...

Keyword(s):

Stem Cell ◽

Nk Cells ◽

Natural Killer ◽

Cytokine Production ◽

Cell Function ◽

Conflicts Of Interest ◽

Cytokine Signaling ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

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Arginine Methylation Of RBM15 By PRMT1 Controls Alternative Splicing Of Genes Involved In Megakaryocyte Differentiation

Blood ◽

10.1182/blood.v122.21.2443.2443 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2443-2443

Author(s):

Xinyang Zhao ◽

Li Zhang ◽

Rui Wang ◽

Ngoc Tung Trans ◽

Hairui Su ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Chromosome Translocation ◽

Arginine Methylation ◽

Mass Spectrometry Analysis ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Spectrometry Analysis ◽

Megakaryocyte Differentiation

Abstract More than 90% of under one year old infants with acute megakaryoblastic leukemia (AMKL) have chromosome translocation t(1;22)(p13;q13) with RBM15 fused to MKL1. RBM15 encodes an RNA binding protein important for hematopoietic stem cell self-renewal and differentiation. In agreement with its roles in AMKL, RBM15 is required for normal megakaryocyte differentiation. We found that higher expression of PRMT1 (Protein Arginine Methyltransferase) is commonly seen in M7 leukemia patient samples than other types of myeloid leukemia and that RBM15 is a bona fide methylation target for PRMT1. Using mass spectrometry analysis, we mapped the PRMT1 mediated mono-methylated site. The enzymatic activity of the PRMT1 V2 isoform is required for RBM15 degradation, as both shRNA molecules knocking down PRMT1 and small chemical PRMT1 inhibitors stabilize the RBM15 protein. Mutation of the methylation site to lysine blocks the ubiquitylation mediated degradation. Thus the degradation is a methylation dependent process. We identified the E3 ligase responsible for the degradation. Down-regulation of the RBM15 protein changes the isoform ratio of genes including GATA1 critical megakaryocyte differentiation. We found that RBM15 regulates its interaction with SF3B1A in methylation dependent manner during alternative splicing of GATA1 pre-mRNA. Thus, via methylation triggered RBM15 degradation, the megakaryocyte progenitor cells maintain a delicate balance between differentiation and proliferation by keeping the proper ratio of GATA1s and GATA1-full length mRNA. SF3B1A has been shown to be mutated in myeloid dysplasia syndrome and in several different types of leukemia. Methylation by PRMT1 links the two types of leukemic genes into a single pathway. Our results imply that targeting PRMT1/RBM15 pathway might be a potential therapy for AMKL and other blood malignancies. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of acute lethal pulmonary inflammation by the IDO–AhR pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615280114 ◽

2017 ◽

Vol 114 (29) ◽

pp. E5881-E5890 ◽

Cited By ~ 16

Author(s):

Soung-Min Lee ◽

Ha Young Park ◽

Young-Sill Suh ◽

Eun Hye Yoon ◽

Juyang Kim ◽

...

Keyword(s):

T Cells ◽

Epithelial Cells ◽

Pulmonary Inflammation ◽

Lung Parenchyma ◽

Lung Epithelial Cells ◽

Dependent Manner ◽

Independent Manner ◽

Hematopoietic Stem ◽

Lung Epithelial ◽

Ifn Γ

The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ–dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ–independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.

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A novel plasma membrane-phillic graphene oxide nanocarrier for neuropeptide delivery to generate tolerogenic dendritic cells in GVHD Immunotherapy

10.21203/rs.3.rs-1148600/v1 ◽

2021 ◽

Author(s):

Xiaohui Wang ◽

Sujing Sun ◽

Chulin He ◽

Chenyan Li ◽

Wenyuan Bai ◽

...

Keyword(s):

Dendritic Cells ◽

Graphene Oxide ◽

Lymph Nodes ◽

Migration Ability ◽

Hematopoietic Stem ◽

Creb Phosphorylation ◽

In Vivo Experiments ◽

Tolerogenic Dendritic Cells ◽

Cytoskeleton Reorganization

Abstract The prevention and the treatment of graft-versus-host disease (GVHD) remains a barrier to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Tolerogenic dendritic cells (TDCs) have become a hotspot in GVHD prevention despite the poor induction efficiency. Herein, we designed a novel size-dependent platform of graphene oxide (GO) nanosheets for neuropeptide delivery for the purpose of TDC generation. GO with a lateral size༞1 µm (L-GO) showed strong affinities to DC membrane, which effectively promoted the recognition between neuropeptide, urocortin (UCN) and its receptor CRHR2 and in turn benefited TDC generation through PKA C/CREB phosphorylation. Simultaneously, L-GO also elevated the expression of CCR7 and enhanced the migration ability of TDCs by mediating cytoskeleton reorganization. In vivo experiments offered direct evidence that TDCs inducted by UCN@L-GO exhibited efficient migration to lymph nodes, abundant generation of Treg, a significant decrease of proinflammatory cytokines and in turn excellent efficiency in GVHD relief. In the current study, we proposed an innovative GO nanosheets based cytomembrane-targeted platform for neuropeptide delivery and subsequent TDC generation. In the meantime, the promoted mobility of TDC pulsed by GOs ensured the high homing efficiency to secondary lymph nodes, attributing to GVHD treatment in vivo. Thus, such work provided a promising strategy that might be applicable more broadly to delivery systems for receptor-mediated drugs, and could meet the changing demand of nanotechnology-based immunotherapy.

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Generation of Retinoic Acid-Dependent Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Blood ◽

10.1182/blood-2020-142468 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-35

Author(s):

Stephanie A Luff ◽

J Philip Creamer ◽

Carissa Dege ◽

Rebecca Scarfò ◽

Samantha Morris ◽

...

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Pluripotent Stem Cells ◽

De Novo ◽

Conflicts Of Interest ◽

Human Pluripotent Stem Cells ◽

Human Embryos ◽

Dependent Manner ◽

Hematopoietic Stem

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic Stem and Progenitor Cells Manifest Direct Migration Towards Secreted DEK in a CXCR2- and G Protein Signaling-Dependent Manner

Blood ◽

10.1182/blood-2019-128738 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1196-1196

Author(s):

Maegan L. Capitano ◽

Yasser Sammour ◽

Maureen Legendre ◽

Scott Cooper ◽

David Markovitz ◽

...

Keyword(s):

Cell Migration ◽

G Protein ◽

Pertussis Toxin ◽

Conflicts Of Interest ◽

Neutrophil Migration ◽

Regulation Of Transcription ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Hematopoietic Stem ◽

G Protein Coupled

DEK, a nuclear DNA-binding protein implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is secreted by macrophages and acts as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK functions as a chemotactic factor attracting neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells; stromal cell-derived factor-1 (SDF-1/CXCL12) being the most potent known protein with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative mouse bone marrow (BM) cells with neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced directed migration of Lin-Sca1+cKit+ (LSK) BM cells at a dose of 100ng/mL, as determined by flow cytometry of input and migrated cells, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ neutrophils. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our recent discovery and report that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8 and binds to the chemokine receptor CXCR2 to regulate hematopoiesis (Capitano et al., 2019, J.C.I. 130: 2555-2570), we hypothesized that DEK may manifest its chemotactic actions through CXCR2, known previously to only bind and mediate the actions of the chemokines IL-8 and MIP-2. To examine this, we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of both LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK cell migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration of LSK cells was observed when IL-8 or MIP-2 was utilized. When Ly6G+ neutrophils were used, CXCR2 neutralizing antibodies blocked migration of the Ly6G+ neutrophils towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ neutrophil migration towards SDF-1. CXCR2 is a G protein-coupled receptor and this interaction can be blocked using pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. To determine if DEK and SDF-1 could inhibit one another, checkboard assays were performed where either different concentrations of DEK was used in the top well and different concentrations of SDF-1 was used on the bottom. Starting at 100ng/mL, DEK in the top well inhibited LSK cell migration towards SDF-1. However, when SDF-1 was in the top well, regardless of the SDF-1 dose, SDF-1 always inhibited LSK cell migration towards DEK. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro but is not as strong of a signaling protein for migration when competing against SDF-1. Thus, DEK may be involved as a compensatory chemotactic agent for HSCs and HPCs, especially under certain inflammatory conditions and when SDF-1 signaling is reduced. Disclosures No relevant conflicts of interest to declare.

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The Ribosome Biogenesis Factor Ltv1 Is Essential for Digestive Organ Development and Definitive Hematopoiesis in Zebrafish

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.704730 ◽

2021 ◽

Vol 9 ◽

Author(s):

Chong Zhang ◽

Rui Huang ◽

Xirui Ma ◽

Jiehui Chen ◽

Xinlu Han ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Target Genes ◽

P53 Protein ◽

Ribosomal Subunit ◽

Dependent Manner ◽

Loss Of Function ◽

Independent Manner ◽

Hematopoietic Stem ◽

Digestive Organs ◽

Stem And Progenitor Cells

Ribosome biogenesis is a fundamental activity in cells. Ribosomal dysfunction underlies a category of diseases called ribosomopathies in humans. The symptomatic characteristics of ribosomopathies often include abnormalities in craniofacial skeletons, digestive organs, and hematopoiesis. Consistently, disruptions of ribosome biogenesis in animals are deleterious to embryonic development with hypoplasia of digestive organs and/or impaired hematopoiesis. In this study, ltv1, a gene involved in the small ribosomal subunit assembly, was knocked out in zebrafish by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated protein 9 (Cas9) technology. The recessive lethal mutation resulted in disrupted ribosome biogenesis, and ltv1Δ14/Δ14 embryos displayed hypoplastic craniofacial cartilage, digestive organs, and hematopoiesis. In addition, we showed that the impaired cell proliferation, instead of apoptosis, led to the defects in exocrine pancreas and hematopoietic stem and progenitor cells (HSPCs) in ltv1Δ14/Δ14 embryos. It was reported that loss of function of genes associated with ribosome biogenesis often caused phenotypes in a P53-dependent manner. In ltv1Δ14/Δ14 embryos, both P53 protein level and the expression of p53 target genes, Δ113p53 and p21, were upregulated. However, knockdown of p53 failed to rescue the phenotypes in ltv1Δ14/Δ14 larvae. Taken together, our data demonstrate that LTV1 ribosome biogenesis factor (Ltv1) plays an essential role in digestive organs and hematopoiesis development in zebrafish in a P53-independent manner.

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Granzyme B Produced by Human Plasmacytoid Dendritic Cells Suppresses T Cell Expansion.

Blood ◽

10.1182/blood.v114.22.2674.2674 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2674-2674

Author(s):

Dorit Fabricius ◽

Angelika Vollmer ◽

Sue Blackwell ◽

Julia Maier ◽

Kai Sontheimer ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Conflicts Of Interest ◽

Granzyme B ◽

Cd40 Ligand ◽

Plasmacytoid Dendritic Cells ◽

Transcriptional Level ◽

Independent Manner ◽

Complex Interactions

Abstract Abstract 2674 Poster Board II-650 Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. In several of these diseases, elevated extracellular levels of the serine protease granzyme B (GrB) are observed. We found that human pDC can be an abundant source of GrB based on FACS analysis, ELISpot, ELISA, Sensizyme, Western immunoblotting, RT-PCR, and fluorescence microscopy. GrB is actively secreted by pDCs and reaches maximal levels up to two logs higher than those produced by classical GrB producers such as CTL or NK cells. However, pDC GrB production is not accompanied by perforin secretion. Spinning disk confocal microscopy revealed that GrB+ pDC bind to and transfer active GrB to T cells. Importantly, this GrB transfer induces a suppression of T cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. GrB expression in pDC is regulated on a transcriptional level by JAK1, STAT3 and STAT5. IL-3 and IL-10 enhance GrB production by pDCs while GrB production is inhibited by toll-like-receptor agonists and CD40 ligand. These findings suggest that GrB production by pDCs is involved in the complex interactions between pDC and T cells and that GrB-secreting pDC may play a regulatory role related to anti-tumor immunity, anti-viral immune responses, and autoimmune processes. Disclosures: No relevant conflicts of interest to declare.

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