Publications by authors named "Axel R Schulz"

13 Publications

  • Page 1 of 1

Deep phenotypical characterization of human CD3 CD56 T cells by mass cytometry.

Eur J Immunol 2021 Mar 15;51(3):672-681. Epub 2020 Dec 15.

Institute for Medical Microbiology and Hospital Hygiene, University of Marburg, Marburg, Germany.

CD56 T cells are a group of pro-inflammatory CD3 lymphocytes with characteristics of natural killer cells, being involved in antimicrobial immune defense. Here, we performed deep phenotypic profiling of CD3 CD56 cells in peripheral blood of normal human donors and individuals sensitized to birch-pollen or/and house dust mite by high-dimensional mass cytometry combined with manual and computational data analysis. A co-regulation between major conventional T-cell subsets and their respective CD3 CD56 cell counterparts appeared restricted to CD8 , MAIT, and TCRγδ T-cell compartments. Interestingly, we find a co-regulation of several CD3 CD56 cell subsets in allergic but not in healthy individuals. Moreover, using FlowSOM, we distinguished a variety of CD56 T-cell phenotypes demonstrating a hitherto underestimated heterogeneity among these cells. The novel CD3 CD56 subset description comprises phenotypes superimposed with naive, memory, type 1, 2, and 17 differentiation stages, in part represented by a phenotypical continuum. Frequencies of two out of 19 CD3 CD56 FlowSOM clusters were significantly diminished in allergic individuals, demonstrating less frequent presence of cells with cytolytic, presumably protective, capacity in these donors consistent with defective expansion or their recruitment to the affected tissue. Our results contribute to defining specific cell populations to be targeted during therapy for allergic conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.202048941DOI Listing
March 2021

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis.

Sci Rep 2019 12 19;9(1):19471. Epub 2019 Dec 19.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7 and IL-6 T cells was detected in early MS-PBMCs, whereas NFAT1T-betCD4 T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7 and MIPβ HLA-DR lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141IRF8CXCR3CD68 dendritic cells. Unlike in Crohn's disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-55852-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923404PMC
December 2019

Stabilizing Antibody Cocktails for Mass Cytometry.

Cytometry A 2019 08 6;95(8):910-916. Epub 2019 May 6.

German Rheumatism Research Center Berlin (DRFZ), a Leibniz Institute, Berlin, Germany.

Mass cytometry is increasingly employed in larger immune profiling studies involving data acquisitions across several days and multiple sites. For gaining a maximum of information from respective data by computational analyses, several techniques have been developed to minimize noise in mass cytometric data sets, such as sample banking, standardized instrument setup, sample barcoding, and signal normalization. However, the repeated preparation of cocktails composed of isotope-tagged antibodies remained a significant source of error. We here show that premixed antibody cocktails fail to deliver expected staining patterns when stored at 4°C for 4 weeks. As a solution, we developed and tested a cryopreservation method for highly multiplexed antibody cocktails for mass cytometry including lanthanide, palladium, and platinum conjugates that yielded stable staining patterns for at least 9 months when stored at temperatures below -80°C. Using frozen aliquots of antibody cocktails is an economic and flexible approach to significantly improve data consistency in large mass cytometry studies with repetitive staining/measurement cycles spanning several days or involving multiple data acquisition sites. © 2019 International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23781DOI Listing
August 2019

Osmium-Labeled Microspheres for Bead-Based Assays in Mass Cytometry.

J Immunol 2019 05 15;202(10):3103-3112. Epub 2019 Apr 15.

German Rheumatism Research Centre Berlin, a Leibniz Institute, 10117 Berlin, Germany; and

Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1801640DOI Listing
May 2019

Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads.

Cytometry A 2019 03 27;95(3):314-322. Epub 2019 Jan 27.

Department of Neurology, Haukeland University Hospital, Bergen, Norway.

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high-parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody-binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry-based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4-integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal-conjugated antibodies for detection of natalizumab and α4-integrin. QSC beads with known antibody binding capacity were stained with the same metal-conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.23723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590231PMC
March 2019

Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry.

Nat Neurosci 2019 01 17;22(1):78-90. Epub 2018 Dec 17.

Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Microglia, the specialized innate immune cells of the CNS, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (10 - 10 cells). We determined expression levels of 57 markers on huMG isolated from up to five different brain regions of nine donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-018-0290-2DOI Listing
January 2019

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

Eur J Immunol 2017 08 18;47(8):1377-1385. Epub 2017 Jul 18.

German Rheumatism Research Center Berlin (DRFZ), Institute of Leibniz Association, Berlin, Germany.

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR labelling protocol. After labelling with Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4 T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue- Nd, CD20-VioGreen- Sm, CD27-Cy5- Er and CD38-Alexa488- Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201747031DOI Listing
August 2017

CD40L expression permits CD8+ T cells to execute immunologic helper functions.

Blood 2013 Jul 29;122(3):405-12. Epub 2013 May 29.

Regenerative Immunology and Aging, Berlin-Brandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany.

CD8(+) T cells play an essential role in immunity against intracellular pathogens, with cytotoxicity being considered their major effector mechanism. However, we here demonstrate that a major part of central and effector memory CD8(+) T cells expresses CD40L, one key molecule for CD4(+) T-cell-mediated help. CD40L(+) CD8(+) T cells are detectable among human antigen-specific immune responses, including pathogens such as influenza and yellow fever virus. CD40L(+) CD8(+) T cells display potent helper functions in vitro and in vivo, such as activation of antigen-presenting cells, and exhibit a cytokine expression signature similar to CD4(+) T cells and unrelated to cytotoxic CD8(+) T cells. The broad occurrence of CD40L(+) CD8(+) T cells in cellular immunity implicates that helper functions are not only executed by major histocompatibility complex (MHC) class II-restricted CD4(+) helper T cells but are also a common feature of MHC class I-restricted CD8(+) T cell responses. Due to their versatile functional capacities, human CD40L(+) CD8(+) T cells are promising candidate cells for immune therapies, particularly when CD4(+) T-cell help or pathogen-associated molecular pattern signals are limited.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2013-02-483586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548794PMC
July 2013

Oligomerization of Uukuniemi virus nucleocapsid protein.

Virol J 2010 Aug 10;7:187. Epub 2010 Aug 10.

Department of Virology, Infection Biology Research Program, Haartman Institute, PO Box 21, University of Helsinki, Helsinki, Finland.

Background: Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail.

Results: A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact alpha-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two alpha-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays.

Conclusions: UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1743-422X-7-187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925374PMC
August 2010

Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Virol J 2010 Jan 15;7. Epub 2010 Jan 15.

University of Bonn Medical Centre, Bonn, Germany.

Background: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.

Results: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.

Conclusions: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1743-422X-7-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2819038PMC
January 2010