Matthias Schiemann - Academia.edu (original) (raw)

Papers by Matthias Schiemann

is online at: Circulation Information about subscribing to Subscriptions:

Translational Medicine Communications, 2020

Background Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are an immature cell ty... more Background Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are an immature cell type that inhibits the effector functions of T lymphocytes in chronic HIV infection. A well-known immunological feature of the disease course is the development of immune exhaustion, which is correlated with excessive immune activation in late-stage disease. Here, we hypothesized that immune exhaustion would also affect PMN-MDSCs in late-stage HIV-1 infection. Methods We evaluated untreated chronically HIV-infected patients (progressors, n = 10) and control groups (controllers, patients with non-small cell lung carcinoma and healthy controls, n = 16) with regard to levels of PMN-MDSCs and their inhibitory potential. Additionally, we studied CD8 T cell effector functions (interferon-gamma, TNF alpha, IL-2 and CD107) and parameters of CD8 T cell activation (CD38 and HLA-DR) and exhaustion (PD-1 and LAG-3) by flow cytometry. Plasma inflammation markers analyzed here were IL-6, IL-8, soluble C...

Cytometry Part A, 2020

Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, ... more Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, temperature, homogenization, buffer composition and mitogens are well known. In contrast, little is known about cell alteration caused by the instrument or the sorting process itself. We systematically evaluated cellular responses to different sorter-induced physical forces. In summary, flow cytometry cell-sorting induced forces can affect cellular signaling cascades, especially the MAPK p38. Functional assays, related to the p38 MAPK pathway, of human primary T cells after flow cytometry sorting did lead to minor physiological modulation but no functional impairments.

Cytometry Part A, 2019

Cyt-Geist REPORT started (WS06). The initiative to embrace and leverage the field of open innovat... more Cyt-Geist REPORT started (WS06). The initiative to embrace and leverage the field of open innovation in cytometry, a new ISAC Open Science group, CYTO Lab Hacks, was launched, with the goal to serve as an open, transparent, and accessible forum for innovation exchange in cytometry (WS07). The use of flow cytometry to generate identifying "fingerprints" of microbial communities was discussed. The awareness of microbial community flow cytometry was raised, and links with practitioners in other fields were established (WS15).

European journal of immunology, Oct 1, 2017

Guidelines for the use of flow cytometry and cell sorting in immunological studies

Aktuelle Dermatologie, 2004

Proceedings of the National Academy of Sciences of the United States of America, 2004

Journal of Visualized Experiments, 2015

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been ... more The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with genespecific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells. Video Link The video component of this article can be found at http://www.jove.com/video/53268/ 14. However, the antibodies might nevertheless adversely affect the cells and each method is restricted to a single pluripotency marker. Finally, fluorescent molecular beacons, dual-antisense oligonucleotides with hairpin structures, which allow live staining of cells, have been described 15. Although this approach may be applied to many genes, the technique requires nucleofection of a single cell suspension, which is not applicable to growing iPS colonies.

The Journal of Immunology, 2015

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response t... more Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220− bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2...

Gastroenterology, Jan 4, 2015

Cancer therapies are being developed based on our ability to direct T cells against tumor antigen... more Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican 3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC) but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. We used mass spectrometry and to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of HLA-A2, and used bioinformatics to identify immunodominant peptides. To circumvent GPC3-tolerance resulting from fetal expression, dendritic cells from HLA-A2 negative donors were co-transfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 mu...

Blood, Jan 24, 2014

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened... more Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentia...

Stem Cells, 2015

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many spe... more The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells c...

Stem cells (Dayton, Ohio), Jan 19, 2014

During cardiogenesis most myocytes arise from cardiac progenitors expressing the transcription fa... more During cardiogenesis most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-over...

Proceedings of the National Academy of Sciences, 2004

Several recent studies have demonstrated that T-helper cell-dependent events during the initial p... more Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8 + T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127 high and CD62L high ) and peripheral effector memory T cells (CD127 high and CD62L low ). Using this new tool of memory T cell analysis, we demonstrate that CD8 + ...

PLoS ONE, 2012

A general obstacle for clinical cell preparations is limited purity, which causes variability in ... more A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve-especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4 high / CD25 high /CD45RA high 'regulatory T cells' and CD8 high /CD62L high /CD45RA neg 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

PLoS ONE, 2013

Adoptive therapy using T cells redirected to target tumor-or infection-associated antigens is a p... more Adoptive therapy using T cells redirected to target tumor-or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3 + T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.

The Journal of Immunology, 2004

Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided c... more Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided cross-presentation of OVA by dendritic cells (DCs). In this study, we analyzed the thesis that CpG-OVA complexes may be cross-presented by B cells to route internalized Ag into the class I MHC presentation pathway. First, we describe that conjugation of CpG-DNA to OVA enhances up to 40-fold internalization of OVA by B cells, which in turn generate the CD8 T cell epitope SIINFEKL complexed to MHC class I, albeit less efficiently than DCs. Furthermore, upon internalization, CpG-DNA conjugated to OVA stimulates B cells to up-regulate costimulatory molecules and cytokines including IL-12. Adoptive transfer of CpG-OVA complex-loaded wild-type B cells cross-primes naive CD8 T cells both in wild-type mice and in MyD88-deficient mice. Overall, these findings disclose attributes of B cells, including cross-presentation of exogenous Ag and cross-priming of naive CD8 T cells that hitherto have been ...

The Journal of Immunology, 2005

Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. I... more Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. In this study, we show that GR1+/CD45+ polymorphonuclear neutrophils (PMN) surprisingly increase the bacterial load of C. pneumoniae in vivo. Upon intranasal infection of wild-type mice, the lung weight is increased; the cytokines TNF, IL-12p40, and IFN-γ, as well as the chemokines keratinocyte-derived chemokine, MCP-1, and MIP-2 are secreted; and GR1+/CD45+ PMN are recruited into lungs 3 days postinfection. In contrast, in infected MyD88-deficient mice, which lack a key adaptor molecule in the signaling cascade of TLRs and IL-1R family members, the increase of the lung weight is attenuated, and from the analyzed cyto- and chemokines, only IL-12p40 is detectable. Upon infection, almost no influx of inflammatory cells into lungs of MyD88-deficient mice can be observed. Six days postinfection, however, MyD88-deficient mice were able to produce TNF, IFN-γ, keratinocyte-derived chemokine, and...

Journal of Clinical Investigation, 2004

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thoug... more Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra-and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-α and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Immunity, 2005

The identification of specific genetic loci that contrib-Ulrike Huffstadt, 1 Andreas Schröder, 1,... more The identification of specific genetic loci that contrib-Ulrike Huffstadt, 1 Andreas Schröder, 1,11 ute to inflammatory and autoimmune diseases has Neil P. Jones, 3 Thomas Peters, 1 Helmut Fuchs, 5 proved difficult due to the contribution of multiple in-Martin Hrabe de Angelis, 5 Michael Nehls, 1 teracting genes, the inherent genetic heterogeneity Johannes Grosse, 1 Philipp Wabnitz, 1 present in human populations, and a lack of new Thomas P.H. Meyer, 1,12 Kei Yasuda, 2 mouse mutants. By using N-ethyl-N-nitrosourea (ENU) Matthias Schiemann, 2,7 mutagenesis to discover new immune regulators, we Christian Schneider-Fresenius, 1,13 Wolfgang Jagla, 1 identified a point mutation in the murine phospholi-Andreas Russ, 8 Andreas Popp, 1 Michelle Josephs, 3 pase Cg2 (Plcg2) gene that leads to severe spontane-Andreas Marquardt, 1 Jürgen Laufs, 1 ous inflammation and autoimmunity. The disease is Carolin Schmittwolf, 1 Hermann Wagner, 2 composed of an autoimmune component mediated by Klaus Pfeffer, 2,14 and Geert C. Mudde 1,15 autoantibody immune complexes and B and T cell in-1 Ingenium Pharmaceuticals AG dependent inflammation. The underlying mechanism Fraunhoferstrasse 13 is a gain-of-function mutation in Plcg2, which leads 82152 Martinsried, Munich to hyperreactive external calcium entry in B cells and Germany expansion of innate inflammatory cells. This mutant 2 Institute of Medical Microbiology, Immunology identifies Plcg2 as a key regulator in an autoimmune and Hygiene and inflammatory disease mediated by B cells and Technical University Munich non-B, non-T haematopoietic cells and emphasizes Clinic Rechts der Isar that by distinct genetic modulation, a single point mu-Trogerstrasse 4a tation can lead to a complex immunological phe-81675 Munich notype. Germany 3 Cancer Research UK Centre for Cell Introduction and Molecular Biology Chester Beatty Laboratories Spontaneous inflammation and autoimmunity occur to-The Institute of Cancer Research gether in diseases such as systemic lupus erythema-Fulham Road tosus (SLE), rheumatoid arthritis, and Wegener's granu-London SW3 6JB lomatosis (WG) (Sack and Fye, 2001). Indeed, the United Kingdom association is so tight that it is not known whether an 4 Institute of Pathology unrestrained inflammatory response or an inappropri-5 Institute of Experimental Genetics ate autoimmune response to self-antigen or both initi-GSF-National Research Center for Environment ate the diseases. Molecular mechanisms involved are and Health further obscured by the complex genetics underlying Ingolstädter Landstrasse 1 this group of diseases that make identification of key 85764 Neuherberg regulatory genes difficult (Roberton and Vyse,