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  • American Society of Hematology  (33)
  • 1
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    Interplay Between the Immune System and Colorectal Carcinoma - Towards Tumor-Specific Peptide-Based Vaccination for Any HLA-Type
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 1027-1027
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1027-1027
    Abstract: Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing. Methods: The HLA presented immunopeptidome of 30 primary CRC samples and matched autologous non-malignant colon tissue was analyzed after HLA-immunoprecipitation by uHPLC tandem mass spectrometry. Identified source proteins and peptides were cross-evaluated with an in-house database of ligandome data derived from different benign tissues to preclude off-tumor presentation of HLA-ligands in order to to prevent any possible induction of autoimmunity by antigen-specific T cells against these ligands. Furthermore, we evaluated pre-existing antigen-specific T cell responses against these novel CRC-associated peptides in a cohort of 25 CRC patients. Results: About 17,000 MHC class I presented peptides could be identified on CRC and were cross-evaluated with our in-house database containing 39,000 MHC class I peptides presented on non-malignant tissue. The ligands identified were derived from 7,500 unique tumor-associated proteins and from 11,500 tumor-exclusive source proteins, respectively. Moreover, about 2,000 different MHC class II presented peptides from more than 1,600 source proteins were identified on CRC tissue. Interestingly, MHC class II peptides could contain shorter amino-acid sequences which - after further proteasomal degradation - might also bind to MHC class I molecules. For clinical applicability, we prioritized the identified ligands according to their frequencies of presentation and detectability on CRC. For representation in the HLA-specific peptide sets, a cutoff expression on at least 25% of allotype-matched tumors was required. This guided the assembly of a peptide vaccine warehouse consisting of 40 highly specific CRC-associated HLA ligands. Therefore, this selected set of HLA-specific peptides allows vaccination against CRC for about 95% of Caucasian patients. So far, we detected specific T cell reactivity against two peptides (detection of 10-fold and 17-fold increase of IFNγ-producing T cells representing spots as compared to background in enzyme linked immunospot assays - ELISPOT) in CRC patients. Conclusions: We here provide for the first time a comprehensive evaluation of HLA-ligandomes in CRC across the most frequent Caucasian HLA-alleles. We developed a HLA-allotype specific warehouse of newly identified CRC associated HLA-ligands, which can be individually assembled according to patient specific HLA-alleles. This approach expands the applicability of peptide-based cancer vaccines to literally every CRC patient. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.1027.1027
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
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  • 2
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    Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling
    American Society of Hematology ; 2013
    In:  Blood Vol. 121, No. 5 ( 2013-01-31), p. 723-733
    Details
    In: Blood, American Society of Hematology, Vol. 121, No. 5 ( 2013-01-31), p. 723-733
    Abstract: Steady-state and emergency granulopoiesis are both dependent on TLR signaling.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2012-05-429589
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
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  • 3
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    Unique Alterations in the Immunopeptidome of Colorectal Cancer Reflect Specific Transformations in Cancer-Associated Signaling Pathways and Reveal Tumor-Specific HLA-Ligand Modulations
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 862-862
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 862-862
    Abstract: Background: Immunotherapy with checkpoint-inhibitors has shown spectacular results in the treatment of certain cancer types including microsatellite instable colorectal cancer (CRC). Applicability is believed to be dependent on the number of potential neo-epitopes derived from genetic mutations that are presented on cancer cells. In case of most microsatellite-stable CRC however, clinical responses to immune checkpoint blockade are so far disappointing. Therefore, we analyzed the non-mutant HLA immunopeptidome of CRC in order to provide an extensive dataset for the development of immunotherapeutic strategies in this very common malignancy. Methods: Tissue specimens from 35 primary CRC and corresponding non-malignant colon were analyzed after HLA immunoprecipitation by uHPLC tandem mass spectrometry. Maximally attainable quantities of source proteins (MAQS) expectable in HLA-ligandomes were estimated by regression analyses. Identified peptides and source proteins were annotated for their pathway association using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the protein analysis through evolutionary relationships (PANTHER). HLA-ligands and source proteins were further analyzed semi-quantitatively assessing significant modulations on CRC tissue compared to adjacent benign colon, with particular focus on specific alterations observed exclusively in CRC tissue. Immunogenicity of the identified HLA-ligands (prioritized ligands for the 8 most frequent HLA-alleles HLA A*01, A*02, A*03, A*24, B*07, B*08, B*44, C*07) was evaluated in peripheral blood mononuclear cells (PBMC) from 50 additional CRC patients and 120 healthy controls (15 each for the 8 most frequent HLA-types) using ELISpot and flow cytometry. Results: For MHC class I, peptides from 7684 source proteins (81% MAQS) were identified on CRC, as well as peptides from 6312 source proteins on (non-malignant) colon tissue (79% MAQS). For MHC class II, peptides from 1602 source proteins (63% MAQS) were identified, as well as peptides from 3835 source proteins on non-malignant colon tissue (75% MAQS). HLA-ligands and their respective source proteins were compared on tissue level (CRC vs. adjacent benign tissue), as well as against a database of 100 non-malignant human tissues of different origin in order to identify ligands and source proteins exclusively presented on CRC tissue. Implementing KEGG and PANTHER pathway analysis, overrepresented source proteins within MHC class I and II restricted ligands could be assigned to classical tumorigenesis pathways like WNT- and integrin signaling, as well as to the p53 signaling pathway. HLA ligands were further semi-quantitatively analyzed comparing tumor and autologous adjacent colon tissue leading to the exclusive identification of 1364 up-modulated and 1070 down-modulated source proteins in CRC tissue. Notably, 3 source proteins (represented by 10 HLA-ligands) showing significant up-modulation and frequent tumor-exclusive detection of derived HLA-ligands (in ≥3 CRC) were identified (LAMC-2, SLC52A2, SULF-1). For MHC class II, a single up-modulated source protein with exclusive detection in ≥2 CRC tissues (IL6R) was identified. Further analyses of HLA class I restricted peptides (n=359) derived from simultaneously up- and down-modulated source proteins revealed that the respective modulation was mainly a peptide sequence specific feature (31/359 (8.6%) peptides with up- and down-modulation). Preexisting T cell responses were observed against one tumor-exclusive up-modulated peptide (SULF-1) in CRC patients and 3 HLA restricted peptides established as immunogenic epitopes in CRC patients (TACC2, TNS4, IGHG2), as well as 2 additional HLA-restricted peptides confirmed as epitopes in healthy controls (GLA, ESRRA). Conclusions: We provide the first comprehensive analysis of the HLA immunopeptidome in a solid cancer (CRC). We observed that the presented ligandome can reflect tumor-specific alterations in protein metabolism. Moreover, tumor-exclusive up- and down-modulation of HLA-peptides was mainly sequence-specific, suggesting a differential posttranslational regulation of HLA-restricted peptides in CRC. The described approach for identification of relevant antigens might also enable patient-specific immunotherapeutic approaches in CRC patients. Disclosures Kowalewski: Immatics Biotechnologies GmbH: Employment. Bernhardt:DECODON: Employment.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.862.862
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
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  • 4
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    Mesenchymal Stem Cell Differentiation Markers Shared with Soft Tissue Sarcomas
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 4755-4755
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 4755-4755
    Abstract: Malignant tumors are hypothesized to harbor small populations of self-renewing cancer stem cells. Targeting these cells may be the decisive step to overcome treatment resistance and achieve tumor eradication in cancer patients. Advanced soft tissue sarcomas (STS) are rare tumors with a dismal prognosis and a small number of systemic treatment options. STS may originate from mesenchymal stem cells (MSC); the latter have mainly been isolated from adult bone marrow (BM) as non-hematopoietic, self-renewing cells whose in vitro progeny comprises osteoblasts, chondroblasts, myocytes, and adipocytes. While in vitro expression profiles of MSC have been investigated extensively, the in vivo counterparts of MSC are still hypothetical. To target rare human cell BM populations including MSC, an exclusive antibody panel was developed. The target antigens include platelet-derived growth factor receptor-β (CD140b), HER-2/erbB2 (CD340), the recently described W8B2 antigen as well as several surface antigens identified by novel antibodies. To define the expression pattern of MSC-markers in STS, three STS cell lines were tested for expression of these antigens. In addition, snap-frozen primary STS sections were analyzed by immunohistochemistry using the same antibody panel. All cell lines revealed expression of selected markers including CD340, W8B2, and CD140b. Several MSC markers were restricted to a subpopulation of cells. In addition, leiomyosarcoma cells displayed a different expression pattern as compared to liposarcoma and Ewing’s sarcoma cells. Results of immunohistochemistry analysis of primary leiomyosarcoma tumor samples correlated strongly with expression patterns established by FACS analysis. However, important cytoarchitectural features regarding selected markers were revealed by immunohistochemistry: while primary leiomyosarcomas displayed uniform expression of W7C6, HEK3D6, CD10, and CD318, other markers such as CD34, W5C5, and 57D2 were expressed by tumor endothelia only. Moreover, a population of perivascular tumor cells was found to express the MSC-markers W4A5, W8B2, CD140b, W3D5, and W5C4. Novel MSC-markers are expressed by subpopulations in STS cell lines as well as in primary sarcoma tissue. Further studies on the functional significance of these phenotypical studies are underway and may help to identify novel specific targets recognizing the self-renewing STS-compartment.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.4755.4755
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
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  • 5
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    Knock-Down of the NFAT2 Long and Intermediate Isoforms Leads to CLL Acceleration
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 4371-4371
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 4371-4371
    Abstract: NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation. We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice. While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells. In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.4371.4371
    RVK:
    XA 33000
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
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  • 6
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    Commensal Bacteria Are Dispensable in Neutropenia-Induced G-CSF Regulation
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 4830-4830
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 4830-4830
    Abstract: Abstract 4830 Introduction: G-CSF is generally accepted to be the major cytokine regulating neutrophil production, but neutrophil steady-state homeostasis has still not been fully elucidated. We have previously shown in NODSCIDcγ−/− mice that feedback G-CSF expression upon antibody-based neutrophil depletion occurs independent from lymphocytes (Bugl et al., ASH 2010) and hypothesized that there may be a sensing mechanism of neutropenia relying on the presence of microbial components, similar to emergency granulopoiesis. Therefore, germ-free wildtype mice received anti-neutrophil antibodies and underwent further analysis. Moreover, passive regulation of circulating G-CSF levels by neutrophil cell mass was examined. Methods: Anti-Ly6-G antibody (clone 1A8) was used to induce neutropenia in germ-free C57BL/6 mice. After one week of neutrophil depletion hematopoietic tissues and peripheral blood were harvested and analyzed on cellular, protein and RNA level. Moreover, neutrophil granulocytes (granulocyte-differentiation antigen-1+ Mac-1+) purified from peripheral blood of C57BL/6 mice were transfused into neutropenic wild type mice and plasma G-CSF was monitored. Results: Peripheral blood neutropenia could be effectively induced in all experimental mice by anti-1A8 antibody. Transfusion of 3.5 × 106 neutrophils into neutropenic wild type mice did not significantly change plasma G-CSF levels. Filgrastim (rhG-CSF), however, caused significant downregulation of bone marrow G-CSF at the mRNA level. Germ-free C57BL/6 mice were analyzed after 1 week of antibody-induced neutropenia and showed a shift of myeloid progenitors towards granulocyte macrophage precursors (GMP) at the expense of megakaryocyte erythrocyte progenitors (MEP) as well as significantly increased numbers of hematopoietic stem cells. In addition, G-CSF, M-CSF, and CXCL12 behaved identically in both germ-free control and wild type mice under specific-pathogen free conditions. Conclusions: Transfusion of G-CSF-receptor (CSF3R) positive neutrophils did not significantly influence G-CSF. Moreover, exogenous G-CSF downregulated marrow G-CSF on the transcriptional level. Germ-free mice are able to mount a feedback loop including G-CSF upregulation and marrow changes in progenitor cell distribution analogous to mice carrying physiological commensal bacteria. Our data are therefore consistent with G-CSF feedback regulation occurring independent from commensal germs. Moreover, our data indicate transcriptional rather than CSF3R+ cell mass-associated passive regulation of G-CSF levels. Taken together, we propose a model of myeloid bone marrow homeostasis, where feedback loops of neutrophil production upon antibody-dependent neutropenia occur through transcriptional upregulation of G-CSF. The underlying mechanisms occur independent of lymphocytes and presence of germs. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.4830.4830
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
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  • 7
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    Transfer of MHC Class I by Coating Platelets Enables Tumor Cells to Evade NK Cell Immune Surveillance by Conferring a “Pseudo Self” Phenotype
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 583-583
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 583-583
    Abstract: Abstract 583 NK cells are a central component of the cytotoxic lymphocyte compartment capable of lysing tumor cells without prior immune sensitization of the host. The mechanisms leading to activation of NK reactivity are described by the principles of ‘missing-self' and ‘induced-self', which imply that cells with a low or absent expression of MHC class I (‘missing-self') and/or a stress-induced expression of ligands of activating NK receptors like e.g. NKG2D (‘induced-self') are preferentially recognized and eliminated by NK cells. Thus, a balance of various activating and inhibitory signals determines whether NK cell responses are initiated or not. Tumor cells often downregulate expression of MHC class I to evade T cell-mediated immune surveillance, which results in enhanced NK susceptibility. Besides the direct interaction with their target cells, NK activity is further influenced by the reciprocal interplay with various other hematopoietic cells. We and others demonstrated previously that thrombocytopenia inhibits metastasis in murine models, which is reversed by additional depletion of NK cells (e.g., Jin et al., Nature Med. 2006, Palumbo et al., Blood 2005). However, the mechanisms by which platelets impair NK-tumor interaction are largely unclear, especially in humans. Recently we reported that platelets release TGF-β upon interaction with tumor cells causing downregulation of NKG2D on NK cells, which impairs anti-tumor immunity by disturbing the principle of “induced self” (Kopp et al., Cancer Res. 2009). Here we demonstrate that platelets further enable tumor cells to evade NK cell immune surveillance by preventing detection of “missing self”: We found that tumor cells rapidly get coated in the presence of platelets, the latter expressing large amounts of MHC class I on their surface. In case of MHC class I-negative or -low cancer cells, this process results in MHC class I “pseudoexpression” on the tumor cell surface as revealed by flow cytometry, immunofluorescent staining, and electron microscopy. Platelet-derived MHC class I was found to inhibit the reactivity of autologous NK, both upon activation with cytokines and, most importantly, in cultures with platelet-coated tumor cells. Using constitutively MHC class I-negative/low tumor cells we found that blocking MHC class I restored NK cytotoxicity and IFN-γ production against platelet-coated tumor cells, but did not alter NK reactivity against the tumor cells in the absence of coating platelets. Taken together, our data indicate that platelets enable a molecular mimicry of tumor cells, allowing the latter to downregulate MHC class I in order to escape T cell immunity without inducing sufficient NK tumor immune surveillance due to conferred platelet-mediated “pseudo self”. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.583.583
    RVK:
    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
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  • 8
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    C-CBL Is a Critical Negative Regulator of Megakaryopoesis
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 1581-1581
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1581-1581
    Abstract: Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p 〈 0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.1581.1581
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 9
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    Lymphocytes Are Dispensable In Neutrophil Homeostasis.
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2613-2613
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2613-2613
    Abstract: Abstract 2613 Introduction: In contrast to red cell or platelet homeostasis, the feedback mechanisms involved in the control of peripheral neutrophil numbers are incompletely understood. Granulocyte-Colony Stimulating Factor (G-CSF) is generally accepted to be the most important determinant of neutrophil production and release from the bone marrow. Recently, a feedback loop including a specialized subset of T-lymphocytes (Tn cells) has been established to explain the correlation between peripheral neutrophil clearance and increased G-CSF production. Methods: Wild type C57bl/6 mice or NODSCIDcγ−/− received anti-Gr1 or anti-1A8 antibodies to deplete neutrophils. Hematopoietic tissues and peripheral blood were harvested at different times and analyzed on cellular, protein and RNA level. Results: Both anti-Gr1 and 1A8 antibodies reduced neutrophils effectively and persistently in vivo. The reaction on neutrophil depletion in the marrow uniformely consisted of an absolute increase in lin-/Sca1+/c-kit+ (LSK) cells and a shift of myeloid progenitors towards granulocyte-macrophage precursors (GMP) and common myeloid progenitors (CMP) at the expense of megakaryocyte-erythrocyte precursors (MEP). Of note, exogenous G-CSF resulted in identical changes. We therefore hypothesized that neutrophil depletion increases G-CSF through a feedback regulatory loop. Neutrophil depletion with anti-Gr1 antibody in wt mice increased G-CSF levels up to approximately 8-fold. While previous observations suggest that G-CSF may be passively regulated through receptor binding and internalization by mature neutrophils, we also found a 10-fold increase of G-CSF mRNA in the marrow. These findings are consistent with active feedback. Interestingly, the effects of 1A8 antibody on G-CSF were less pronounced. Instead, mice depleted of neutrophils with 1A8 antibody displayed predominant increases of M-CSF, suggesting redundant feedback pathways. Our results in wildtype mice treated with 1A8 antibody confirm previous data by Stark et al. (Immunity 2005; 22: 285–294), including increases of IL-23 and IL-17 after neutrophil depletion. However, when neutrophils were depleted in NODSCIDcγ−/− mice, who lack lymphocytes and NK-cells, both IL-23 and IL-17 remained unchanged, but G-CSF levels still increased markedly. Conclusions: Effective neutrophil depletion by antibodies directed against different neutrophil antigens uniformly results in major shifts in the hematopoietic bone marrow showing an increase in the number of LSK hematopoietic stem cells accompanied by a significant increase in myeloid progenitors at the expense of thrombopoietic, red cell, and lymphoid precursors. Our results demonstrate regulatory feedback loops of neutrophil granulopoiesis culminating in increased production of myelopoiesis stimulating cytokines such as G-CSF, GM-CSF, and M-CSF. The underlying chain of events includes IL-23 and IL-17 in wild type mice as previously described. However, additional redundant pathways exist in lymphocytopenic NODSCIDcγ−/− mice. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2613.2613
    RVK:
    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
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  • 10
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    Defects in Primary Hemostasis and Acquired Von Willebrand Disease As Cause of Bleeding in Patients with Multiple Myeloma
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 3540-3540
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3540-3540
    Abstract: Introduction. Paraproteinemia has been associated with both thromboembolic and bleeding complications. Previous retrospective analyses and case reports report on a direct correlation of serum immunoglobulin levels (especially IgM and IgA) with critical bleeding events. Underlying mechanisms include interference with platelet function, immune thrombocytopenia, clotting factor deficiencies, and acquired von Willebrand disease (AVWD). Methods. Laboratory data, including qualitative and quantitative analyses of von Willebrand Factor (vWF), collagen binding assay (CBA) and PFA-100 closure time from 132 patients with diagnosis of multiple myeloma between 2003 and 2015 were retrieved, and cases were retrospectively evaluated for bleeding complications. Coagulation abnormalities were correlated with clinical history and disease parameters. Results. 70 out of 132 patients (53%) displayed bleeding complications including multiple hematomas (n=41), epistaxis (n=10), or perioperative bleeding (n=9). Bleeding vs. non-bleeding patients showed no differences regarding age, gender, previous chemotherapy, or immunoglobulin isotypes. Special coagulation lab results of patients with bleeding events are shown in table 1. Table 1. Pathological coag labs in 70 patients with bleeding Normal Abnormal Sensitivity(95% CI) Specificity(95% CI) PPV*(95% CI) NPV*(95% CI) PLT ( 〈 50x103/µl) 70 0 0 1 0 0.47 (0.38-0.56) PT, aPTT 68 2 0.06 (0.02-0.1) 0.95 (0.85-0.99) 0.57 (0.2-0.88) 0.47 (0.38-0.56) vWF:Ag or RCoF 64 6 0.09 (0.04-0.18) 0.95 (0.86-0.99) 0.67 (0.3-0.9) 0.48 (0.39-0.9) PFA-100 27 43 0.61 (0.49-0.73) 0.97 (0.88-0.99) 0.96 (0.84-99) 0.69 (0.58-0.78) CBA/vWF 45 25 0.35 (0.25-0.48) 0.98 (0.9-0.99) 0.96 (0.78-1) 0.58 (0.48-0.67) PFA-100 or CBA/vWF 52 18 0.74 (0.62-0.84) 0.95 (0.86-0.99) 0.94 (0.84-1) 0.76 (0.65-0.85) *PPV = positive predictive value, NPV = negative predictive value Serum free light-chain, but not complete Ig paraprotein levels were significantly associated with bleeding events (p 〈 0.001), prolonged PFA-100 closure time (p 〈 0.001), and pathological CBA/vWF-ratio (p 〈 0.05). Conclusions. Bleeding events were most commonly due to defects of primary hemostasis. Global coagulation tests as well as vWF:Ag and RCo were insufficient in predicting bleeding. When the results of both PFA-100 and CBA/vWF-ratio were considered, 74% of the bleeding patients could be detected. We suggest that both PFA-100 and CBA/vWF-ratio should be determined in patients with multiple myeloma and signs of bleeding in order to rule out AVWD with sufficient sensitivity. Serum free light-chain levels were correlated with hemorrhage and detectable AVWD. Therefore, free Ig light-chains may play a role in the pathophysiology of bleeding. Disclosures Weisel: Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; Noxxon: Consultancy; Onyx: Consultancy, Honoraria; Novartis: Other: Travel Support; Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Support; BMS: Consultancy, Honoraria, Other: Travel Support.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.3540.3540
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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