Suspension MC Session

Experimental and Clinical Research Center (ECRC), Charité – Universitätsmedizin Berlin and the MDC

Unravelling the complexity of human neuroinflammation with omics technologies
Neuroinflammation is a common feature of autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS), in which the pathologies are associated with various immune responses in different body compartments e.g. local inflammation, microglial activation, and CNS infiltration of circulating immune cells. Characterization of more diverse immune cell types residing in different body compartments including peripheral blood, cerebrospinal fluid (CSF), gut, brain interface and brain parenchyma is required for better understanding dynamic compartmentalization of these cells in early as well as in the progressive stages of diseases. Apart from immune responses, evaluating changes in metabolome and protein expression profiles of tissue and fluidic compartment and investigating how these changes associate with functional and phenotypic changes of immune cells in different body compartments will provide us more insights in dynamic cell signalling of immune cells from/between different compartments towards the CNS and will further unravel neuroinflammation-associated phenotypic/functional transmission of immune cells and how they are involved in CNS homeostasis and disease progression/severity.

Biosketch
Chotima Böttcher is a research team leader of the Clinical Neuroimmunology group at the Experimental and Clinical Research Center (ECRC), Charité – Universitätsmedizin Berlin and the MDC, Germany. Dr. Böttcher obtained her PhD at Institute of Pharmacy, Faculty of Natural Sciences at Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany. In the first year of her postdocteral fellow, she continued her study on biosynthesis of mammalian morphine at Martin-Luther-University Halle-Wittenberg and later at Donald Danforth Plant Science Center, MO, USA. In 2006, she moved to Charité – Universitätsmedizin Berlin and has started her new research field –systems immunology in neuroscience, with particular emphasis on myeloid cells including monocytes and brain microglia/macrophages. Currently, her research aims to identify cellular and molecular complexity of human myeloid cells in different body compartments during neuroinflammation.

Deutsches Rheuma-Forschungszentrum Berlin, a Leibniz-Institute

Identifying baseline predictors of vaccination outcome in peripheral blood and gut microbiota

Vaccination effectively protects from severe consequences of infection. However, little is known about immunological determinants of vaccination success in humans. We deeply profiled peripheral blood leukocytes by mass cytometry in cohorts of older (>80 years, n=55) and younger adults (20–53 years, n=44) before receiving at least two doses of BNT162b2 mRNA vaccine and correlated the data with the SARS-CoV-2-specific response data. Vaccination responses expectedly varied stronger among older compared to younger individuals, including older individuals with nearly no detectable T- and B-cell responses, as described in previous studies.
Our mass cytometry data reproduced known features of immune ageing in senior adults. Additionally, we identified signatures of high and low responsiveness among senior vaccinees. Older individuals with high antibody responses were characterized, by increased frequencies of pro-inflammatory, intermediate CD16+CD14++ and non-classical CD16+CD14+/- monocytes as well as pro-inflammatory CD38+CD11c+ NK cells. This suggests an unexpected beneficial role of these immune subsets for the vaccination response in elderly vaccinees, whereas they are usually considered detrimental to vaccine responsiveness in young individuals. In contrast, older subjects with low antibody response showed fewer transitional CD38+ naive B cells and more early immature neutrophils in the blood, most likely reflecting an imbalance in Neutrophil-Lymphocyte-Ratio (NLR).
Furthermore, we integrate immune cell profiles with gut microbiota phenotypes from the same cohorts analyzed by multi-parametric bacterial flow cytometry. Using correlation analysis and multi-omic factor analysis (MOFA), we investigate the association of immune cell subpopulations, and their activation status, with specific gut microbial phenotypes. This multi-modal integration is aimed to undercover immune mechanisms of clinical relevance and increases the predictive accuracy on vaccination outcome.
Altogether, we here report baseline immune and microbial predictive signatures associated with the success of mRNA vaccination in senior adults that can be relevant in the clinical practice.

Center for Regenerative Therapies TU Dresden (CRTD), Dresden, Germany

Deciphering the epigene,c state of neural cell types during neocortex
development

The neocortex is the most recently evolved brain structure and aHributed to higher cogni<ve
func<ons. The development of the neocortex is governed by spa<al and temporal gene
expression programs, which are regulated by transcrip<on factors and epigene<c
mechanisms, such as posHransla<onal modifica<ons of histone proteins. Specific histone
modifica<ons act on genomic loci to repress or promote gene expression, thereby
contribu<ng to tuning prolifera<on and differen<a<on of neural progenitor cells. The balance
between these processes is key to control neocortex size. Misregula<on of neocortex
development oMen results in neurodevelopmental disorders, which may include changes in
the brain size, oMen linked to intellectual disability. Given the large number of epigene<c
modifica<ons, we have a limited understanding of epigene<c state in different neural cell
popula<ons and the temporal changes in specific histone modifica<ons in the developing
human neocortex. To unravel the epigene<c state during brain development, we have
established an epigene<cs core panel for CyTOF to provide the single cell informa<on on more
than thirty epigene<c readouts in combina<on with ten neural cell type markers. To model
human neocortex development, we generate human cor<cal organoids from induced
pluripotent stem cells. Our preliminary Epi-CyTOF data suggests dis<nct epigene<c marking in
neural progenitor cells and neurons. Altogether, Epi-CyTOF presents a powerful technology to
elucidate the complexity and dynamics of a large panel of histone modifica<ons during human
neocortex development.

Deutsches Rheuma-Forschungszentrum Berlin, a Leibniz Institute
In-Depth Characterization of Antigen-Specific Human B Cells by High-Dimensional Mass Cytometry

Studying B cells at the level of antigen-specificity is inevitable to understand the principles of memory induction and maintenance in the B-cell lineage. We here present a new method for the high-avidity detection of antigen-specific human B cells by mass cytometry. This novel approach employs a synergistic combination of biotin-labeled antigens with isotope-labelled streptavidin, commonly referred to as tetramers.

Our B-cell panel allows the simultaneous assessment of several B-cell specificities, such as SARS-CoV-2 RBD and NP, utilizing a combinatorial tetramer approach. The comprehensive characterization of B cells is achieved through the incorporation of additional markers targeting various surface, intracellular, and immunoglobulin molecules. The approach is compatible with both live and fixed cells, and it facilitates advanced techniques such as sample barcoding (up to 20-plex) and pre-enrichment of the B cells using the MACSQuant Tyto Cell Sorter, ensuring maximum sample standardization and throughput.

The versatility of our panel provides a powerful platform for the investigation of various antigen-specific B-cell subsets in a single assay, even with limited sample sizes. The method holds promise for diverse contexts, including autoimmune research e.g. multiple sclerosis (MS), and the investigation of immune responses to vaccinations or infections.

Center for Regenerative Therapies Dresden
Elucidating neonatal hematopoietic stem cells specific pathways in maternal Type 1 Diabetes using mass cytometry

Type 1 Diabetes (T1D) is an autoimmune disorder in which pancreatic ß-cells are targeted by immune cells leading to insulin deficiency. Offspring of T1D mothers showcase significantly reduced risk of developing T1D than offspring of T1D fathers. Unpublished data from our lab suggests an increase in a specific hematopoietic stem- and progenitor cell (HSPC) compartment in umbilical cord blood (UCB) from offspring of T1D mothers at birth. Here we aimed to elucidate the mechanisms that underly this observation by mimicking the diabetic gestational environment in-vitro, using UCB as a HSPC source. Therefore, we established an antibody panel comprising 28 extracellular- and 12 intracellular markers to quantify HSPC populations and phosphorylation dependent signaling pathways by mass cytometry powered by Time of Flight (CyTOF, Standard BioTools). To optimize the panel we titrated all antibodies, configured settings and standardized a staining protocol. The robustness and efficacy of the panel was then evaluated on CD235a-depleted, CD34+ (HSPCs) enriched CB cells with CyTOF2 followed by high-dimensional single – cell analysis using the OMIQ software. Using the panel, we were able to detect HSPC cell populations and define populations with upregulated phosphorylation after short term stimulation with cytokines including IL-3 and GM-CSF. We observed upregulated STAT3 and ERK signaling in HSPCs, monocytes and dendritic cells, a sign of induced proliferation and stress response. Furthermore, signal intensities for lineage markers across samples were comparable indicating panel stability and data replicability. Thus, with this approach, we can detect how HSPCs as well as other cord blood cellular populations respond to a defined cytokine stimulation. The impact of parameters present during gestation in mothers with T1D on HSPC response will be tested in future experiments, hopefully helping to understand how the intrauterine environment imprints offspring from mothers with T1D.