bluuebunny/crossref_metadata_2025_split huggingface dataset:
Granzyme B Is a Key Regulator of Plasmacytoid Dendritic Cell Immunogenicity
Authors: Jahrsdörfer Bernd , Beyer Thamara , Schrezenmeier Hubert , Debatin Klaus-Michael , Fabricius Dorit 
Published: October 2019

Abstract
Despite their low frequency of 0,1 - 0,5% in the peripheral blood, human plasmacytoid dendritic cells (pDC) are important modulators of adaptive immune responses and have meanwhile taken the step from the bench to promising initial tumor vaccination studies. PDC express high levels of the IL-3-receptor CD123, MHC class II and blood dendritic cell antigens (BDCA)-2 and -4. Apart from secretion of IFN-alpha and their capacity to rapidly initiate antigen-specific CD8+ T cell responses by cross-presentation, they produce large amounts of the serine protease granzyme B (GrB). GrB exhibits various non-cytotoxic functions including T cell regulation and the support of antigen processing and phagocytosis. We therefore hypothesized that pDC may use their GrB for both T cell regulation and for antigen uptake and processing. PDC were isolated using magnetically labeled antibodies against CD123, and cultured in the presence of various stimuli. Here, we demonstrate that pDC are potent producers of enzymatically active GrB, which reached maximal concentrations up to two logs higher than those produced by cytotoxic cells including CTL and NK cells. The strongest inducers of GrB in pDC were IL-3 and IL-10, whereas toll-like receptor (TLR)-7 or TLR-9 ligands such as CpG ODN or imiquimod as well as CD40 ligand suppressed GrB induction. We demonstrate that pDC-derived GrB can suppress effector T cell proliferation by GrB-mediated degradation of the T cell receptor zeta-chain. Moreover, we tested the uptake of fluorescently labeled antigenic material into pDC after lysis of tumor cells by freeze-thaw cycles and irradiation. These studies revealed a correlation between pDC GrB expression and antigen uptake, with GrBhigh pDC taking up significantly more antigenic material than GrBlow pDC. Our data support a novel concept, in which pDC acquire their maximal immunogenic potential in a two-step activation process: First, high levels of GrB are induced by IL-3 and IL-10, thereby supporting antigen processing and uptake into pDC, while preventing precocious activation of T cells. Subsequently, TLR agonists and CD40 ligand turn off GrB in pDC and initiate the transfer of MHC-antigen complexes and co-stimulatory molecules to the pDC surface, resulting in highly immunogenic pDC. Our data may be of translational relevance for the further development of novel DC vaccination strategies in tumor therapy.

Disclosures
No relevant conflicts of interest to declare.
Interleukin-7 Represses the Immunoregulatory Function of Human TCRαβ+ CD4- CD8- Double-Negative (DN) T Cells By Activating Akt/mTOR Signaling
Authors: Allgaeuer Andrea , Gerbitz Armin , Mackensen Andreas , Voelkl Simon 
Published: October 2019

Abstract
Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). Recently, a novel subset of TCRαβ+ CD4- CD8- (double negative, DN) T cells has been described to possess a very potent suppressive activity on allogeneic T-cell responses in both mice and humans. Of interest, clinical studies in patients after allogeneic stem cell transplantation (SCT) revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. Interleukin (IL)-7 is required for survival and homeostasis of mature T-cells and has been shown to modulate the functional activity of Treg cells. Here we asked whether IL-7 affects the suppressive activity of human DN T cells. We found that human DN T cells isolated from peripheral blood lymphocytes do express the IL-7 receptor and are able to transduce IL-7 downstream signaling. Intriguingly, IL-7 significantly diminished the suppressive activity of DN T cells towards allogeneic CD4+ and CD8+ T cells. Thereby, IL-7 did not render the responder T cells insensitive to suppression. Instead, blocking of key signaling molecules revealed that IL-7 has a direct impact on the suppressive activity of DN T cells. Further analyses demonstrated that IL-7 activates Akt/mTOR signaling in DN T cells. Importantly, selective inhibition of the Akt or mTOR protein kinase was able to reverse the IL-7 effect thereby restoring the suppressive function of DN T cells, whereas inhibition of other central T-cell signaling pathways did not. Thus, DN T-cell mediated suppression is impaired by IL-7 due to crucial activation of the Akt/mTOR signaling cascade. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T-cell functionality in GvHD.

Disclosures
No relevant conflicts of interest to declare.
Platelets Protect Against Murine Pneumonia
Authors: Feng Shuju , Evans Scott , Wu Jessica , Afshar-Kharghan Vahid , Kroll Michael H. 
Published: October 2019

Abstract
Platelets have been called ancient granulocytes because they are capable of recognizing and destroying microbial invaders through their release of a variety of anti-microbial proteins. Experimental evidence indicates that this may involve direct platelet-microbial interactions or occur indirectly through platelet-leukocyte interactions, neutrophil histone-platelet interactions and/or platelet-directed neutrophil extracellular trap formation. To test the hypothesis that platelets contribute to innate lung immunity, we examined the clinical course, lung histopathology and pathogen burden of mice -/+ antibody-mediated platelet depletion who were subjected to aerosolized bacteria (Streptococcus pneumoniae or Pseudomonas aeruginosa). Mice (C57BL/6, 10 weeks) were injected by tail vein with a purified rat monoclonal antibody directed against mouse GpIbα (or control antibody) resulting in platelet depletion to ~ 20% at 3 hours that persisted for 48 hours. 30 minutes after GpIbα antibody injection mice were placed into a closed system for 60 minutes where they were exposed to 10 ml nebulized S. pneumoniae (2-6 x 1011 CFU/ml) or P. aeruginosa (1-4 x 1010 CFU/ml). All platelet depleted animals infected with S. pneumoniae died by day 4 after infection, whereas all platelet replete mice survived the infection. Infection with P. aeruginosa resulted in death of all animals, although the platelet replete mice lived longer than the platelet depleted mice. No platelet depleted sham-infected animals died. The lungs of animals were harvested, fixed and prepared for histopathology with hematoxylin and eosin staining; or harvested, homogenized and plated for bacterial CFU measurements. Whole lungs of platelet-depleted and infected mice were congested. Histopathology showed increased red cells in the venules of platelet-depleted S. pneumoniae-infected mice but no difference in air-space hemorrhage in comparison with platelet-depleted sham-infected lungs and platelet-replete S. pneumoniae-infected lungs. Platelet depletion resulted in ~ 5-fold increase in lung S. pneumoniae CFUs and ~ 10-fold increase in P. aeruginosa CFUs. These results indicate that platelets provide defense against pneumonia. The mechanism of this effect is not known. Experiments are ongoing to determine if it is a consequence of platelet-dependent hemostasis, platelet-leukocyte interactions and/or direct platelet-microbial interactions leading to the release of bactericidal compounds.

Disclosures
Kroll: Boerhinger-Ingelheim: Membership on an entity's Board of Directors or advisory committees; Aplagon Therapeutics: Membership on an entity's Board of Directors or advisory committees.
Melatonin Protects Against Apoptosis of Megakaryocytes Via Its Receptors and Activation of PI3k/Akt Pathway
Authors: Li Su-yi , Zhou Min , Ye Jie-yu , Lian Qi-zhou , Meng Fan-yi , Yang Mo 
Published: October 2019

Abstract
Melatonin has been shown to be able to lower the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherepy in clinical studies (Lissoni et al, J Pineal Res, 1999). Melatonin is an acetylated product of serotonin. Our previous study demonstrated that serotonin stimulates megakaryopoiesis and thrombopoiesis (Yang et al, Stem Cells, 2007; 2014). However, the underlying mechanism of melatonin on platelet formation/survival has not been well investigated. We hypothesized that melatonin has therapeutic effects on thrombocytopenia by reducing apoptosis of megakaryocytes (MKs)/platelets via melatonin receptors and activation of Akt signaling. We investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature looking. Using megakaryocytic cell CHRF-288-11, we identified the expression of melatonin receptors MT2 on these MK cells by RT-PCR and western blotting. We further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt were significantly increased after treatment by melatonin and this effect was abolished by melatonin receptor MT2 blocker. The effect of melatonin on in-vivo thrombopoiesis was also investigated in an irradiated mouse model. Eighteen mice were randomly divided into three groups (6 each). Group 1 (normal control) received no irradiation or melatonin; Group 2 (model control) and Group 3 (melatonin treated) both received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group 3 consecutively for 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.We found that melatonin enhanced the recovery of platelets and WBC production in Group 3. Our results showed that melatonin enhanced platelet formation and survival in radiation induced thrombocytopenia. The underlying mechanisms might involve in direct stimulation of megakaryopoiesis and anti-apoptotic effect on megakaryocytes via it receptors and activation of Akt signaling.

Disclosures
No relevant conflicts of interest to declare.
Thrombin Generation in Zebrafish
Authors: Schurgers Evelien , Moorlag Martijn , Kelchtermans Hilde , Hemker Coenraad , De Laat Bas 
Published: October 2019

Abstract
Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay.
Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm).
Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans.
Table 1. Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments. Experiment 1(n=4) Experiment 2(n=9) Mean CV Mean CV Peak ETP (nM.min) 575.68 19.69 689.71 24.91 Peak (nM) 1573.16 29.58 1668.05 14.46 Lag time (min) 0.27 0.00 0.49 10.92 Time to peak (min) 1.08 5.30 1.37 11.49 Velocity (nM/min) 4002.33 38.21 3826.35 17.04
Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis.

Disclosures
No relevant conflicts of interest to declare.
Small-Scale Mass Spectrometry-Based Phosphoproteomic Analysis of Primary Hematopoietic Stem and Progenitor Cells to Identify Critical Regulators of HSPC Mobilization and Leukemogenesis
Authors: Wang Leo D. , Nguyen Phi , Ficarro Scott B , Hutchinson John , Hofmann Oliver , Marto Jarrod A , Wagers Amy J. 
Published: October 2019

Abstract
Cellular functions are largely effected by proteins, but protein-level analysis of hematopoietic stem and progenitor cell (HSPC) functions has historically been challenged by the difficulty of performing comprehensive and robust proteomic studies of rare cell populations. To confront this challenge, we developed a novel nanoscale multidimensional mass spectrometry-based phosphoproteomic platform that allows, for the first time, comprehensive and unbiased analysis of the activated protein circuits in blood stem cells, as assessed by protein phosphorylation status. We used this platform to interrogate the proteomic features responsible for the growth and maintenance of hematopoietic progenitors. Our analysis pipeline is capable of returning 12,000 unique phosphopeptide sequences (corresponding to several thousand proteins) from an input of 400,000 FACS-sorted primary mouse HSPCs. Among these phosphorylated proteins, the novel Rac-GAP Arhgap25 emerged as an important regulator of mobilization in HSPCs. Arhgap25 is phosphorylated upon treatment of HSPCs with a standard cyclophosphamide-GCSF mobilization protocol. Germline deletion of Arhgap25 in mice impairs HSPC egress from the bone marrow, both at rest and after mobilizing stimuli. These findings validate the use of this platform in the discovery of new therapeutic targets in hematopoiesis, and present a clear pathway for identifying novel targets in other rare subsets of human progenitor cells, including leukemia stem cells.

Disclosures
No relevant conflicts of interest to declare.
PAX5 Tyrosine Phosphorylation By Syk Co-Operatively Works with Its Serine Phosphorylation By ERK1/2 and Cancels PAX5-Dependent Repression of BLIMP1: A Mechanism of Antigen-Triggering Plasma Cell Differentiation through B Cell Receptor Signal
Authors: Inagaki Yuichiro , Hayakawa Fumihiko , Kojima Yuki , Morishita Takanobu , Imoto Naoto , Naoe Tomoki , Kiyoi Hitoshi 
Published: October 2019

Abstract
Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of BLIMP1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of BLIMP1 and XBP-1 and subsequent suppression of PAX5 by BLIMP1 were observed and thought to be the trigger of plasma cell differentiation. We previously demonstrated that serine phosphorylation of PAX5 by ERK1/2, a main component of BCR signal, attenuated the BLIMP1 suppression by PAX5 and that the PAX5 phosphorylation might be the initial event for plasma cell differentiation (Yasuda T et al, J Immunol. 2012; 188: 6127-34). Here, we investigated additional PAX5 phosphorylation by BCR signal and found that another BCR signal component, Syk, caused PAX5 phosphorylation in vitro (Figure A). We identified the tyrosines that were phosphorylated by Syk in vitro by making phosphorylation-defective mutants, and confirmed that Syk phosphorylated PAX5 at the same sites in vivo (Figure B). In the luciferase reporter assays, PAX5 tyrosine phosphorylation by Syk attenuated the BLIMP1 suppression by PAX5, similarly to its serine phosphorylation by ERK1/2, and both phosphorylations co-operatively worked for it (Figure C). Furthermore, we demonstrated that B cell receptor stimulation with anti-IgM antibody induced Syk and ERK1/2 activation, tyrosine and serine phosphorylation of endogenous Pax5, and upregulation of Blimp1 mRNA. These results suggested that PAX5 phosphorylations by Syk and ERK1/2 co-operatively work for the cancelation of transcriptional repression of Blimp1 by PAX5 after BCR activation by antigen. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells.
Figure 1 Figure 1.

Disclosures
Naoe: Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.
Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma
Authors: Botezatu Lacramioara , Al-Matary Yahya S. A. , Opalka Bertram , Dürig Jan , Schroeder Thomas , Haas Rainer , Dührsen Ulrich , Khandanpour Cyrus 
Published: October 2019

Abstract
The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML.
We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes.
Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients.
To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS.

Disclosures
No relevant conflicts of interest to declare.