General Immunotyping
SNP/WGS/WES
The assessment of genetic variation within individuals requires the analysis of variants from the entire allelic spectrum, ranging from common variants that are frequently observed across populations, to variants private to individual pedigrees. Common polymorphisms (i.e., SNPs) are analyzed using array-based genotyping, which is a cost- and time-efficient way to characterize the common individual background across large cohorts. For the analysis of rare variants across protein-coding regions, whole exome sequencing (WES) is widely applied in many research projects. Finally, an individual’s entire genetic variation can be assessed using Whole Genome Sequencing (WGS), which is the most comprehensive assessment of all types of variants across both coding and non-coding regions, respectively. Despite decreasing sequencing costs during the last years, the access to this technology is made difficult as investment in and maintenance of NGS systems as well as the establishment of research specific applications remains expensive and time consuming. At the University Hospital Bonn, WES and WGS applications are enabled by the NGS Core Facility, which is allocated at the Institute of Human Genetics in Bonn.
Kerstin Ludwig (UKB) kerstin.ludwig@uni-bonn.de
Bulk Transcriptomics (PRECISE)
Bulk transcriptomics has emerged over the past decade as a crucial methodology for evaluating the immunological profile of clinical and pre-clinical samples. The PRECISE platform (DZNE) offers a range of bulk transcriptomics approaches and provides expert guidance on selecting the most suitable approach for each sample type. The PRECISE platform can analyze both total RNA (Illumina TruSeq) and enriched mRNA (Illumina mRNA, SMARTseq-2) from diverse sample types, including whole blood (e.g., PAXgene tubes), cerebrospinal fluid, and biobank tissue biopsies. The PRECISE platform also provides state-of-the-art data preprocessing and supports the bioinformatic analysis of individual research projects.
Marc Beyer (DZNE) marc.beyer@dzne.de
Proteome and fluid biomarkers (PRECISE, UKB)
The blood proteome holds immense promise for precision medicine, yet its analysis is hindered by the low abundance of most plasma proteins and the vast dynamic range of protein concentrations. A key technology available at the PRECISE platform is the NUcleic acid Linked Immuno-Sandwich Assay (NULISA). This novel approach enhances the sensitivity of traditional proximity ligation assays by approximately 10,000-fold, achieving attomolar-level detection. This significant improvement is attributed to a dual capture and release mechanism integrated into oligonucleotide-conjugated antibodies, which effectively suppresses assay background. Furthermore, NULISA enables highly multiplexed quantification of both low- and high-abundance proteins across a wide dynamic range. This is accomplished by attenuating signals from abundant targets using unconjugated antibodies and employing next-generation sequencing of barcoded reporter DNA to decode and quantify protein levels.
Marc Beyer (DZNE) marc.beyer@dzne.de
Susanne Schmidt (UKB)
Kerstin Ludwig (UKB)
Anja Schneider (UKB)
High-dimensional cytometry (DZNE, UKB, UBO)
The rapid advancement of high-dimensional cytometry (HDC) methods has revolutionized our ability to analyze millions of cells from thousands of complex tissues. Initially driven by immunological research, where the heterogeneity of cell types and the growing number of cell states significantly benefit from these high-dimensionality techniques, HDC has become an indispensable tool. It now routinely measures up to 50 markers simultaneously at single-cell resolution, making it crucial not only in immunology but also in other disciplines such as microbiology, virology, and neurobiology.
DZNE is equipped with a diverse array of instrument configurations, ranging from conventional flow cytometers (e.g., BD Symphony A5) to cutting-edge spectral cytometers (e.g., BD S8 and BD A5 SE). This combination allows us to perform high-throughput and high-resolution analysis of clinical samples with exceptional precision.
Complementing data acquisition, we provide a modern analytical framework (cyCONDOR) for the comprehensive analysis of high-dimensional data, creating an end-to-end ecosystem for researchers.
Marc Beyer (DZNE) marc.beyer@dzne.de
Lorenzo Bonaguro (DZNE) lorenzo.bonaguro@dzne.de
Natalio Garbi (UKB)
Elvira Mass (UBO)
High Resolution Immunotyping
Single-cell multiomics (PRECISE)
Sequencing-based technologies are pivotal for assessing genomic, transcriptomic, and epigenomic information. Moreover, sophisticated technologies in proteomics, metabolomics, microbiomics, and lipidomics have been integrated into immunological research. In the past decade, single-cell sequencing technologies have emerged, with single-cell transcriptomics spearheading this advancement. The PRECISE platform has established a comprehensive suite of state-of-the-art single-cell technologies, encompassing all major approaches such as 10x Genomics, BD Rhapsody, Parse Bioscience, and Scale Bio, alongside academic protocols like SMART-seq2 and Flash-Seq. The PRECISE platform has also cultivated deep expertise in these technologies, extending to multi-omics experiments where multiple layers of omics information are assessed from each cell, combining up to five independent layers in a single analysis (Gemund et al.). Furthermore, the PRECISE platform provides robust data preprocessing and pre-analysis to facilitate the biological interpretation of the data by the end-user.
Marc Beyer (DZNE) marc.beyer@dzne.de
Mass Spectrometry-based Proteomics
Mass spectrometry (MS)-based proteomics has evolved from cataloging proteins in biological specimens to assessing protein properties and their functional modulation on multiple levels. Modern proteomics approaches interrogate the engagement and reactivity of bioactive molecules, resolve spatio-temporal dynamics in protein signaling networks, and yield comprehensive functional annotation of post-translational modifications (PTMs) on a proteome-wide scale. Among other approaches, nanoparticle-fractionated body fluid proteomics is emerging as a powerful tool as it overcomes the high-dynamic range issue and quantifies over 6000 proteins, including over 40 cytokines in plasma or serum. Due to the unbiased MS-based detection, PTMs (such as phosphorylation or protein cleavage), which are often indicative of biological activities or disease states are captured providing a deeper mechanistic understanding compared to affinity-reagent-based approaches (such as Olink or Somalogic). We expect that this technology will delineate novel diagnostic, prognostic biomarkers for patient stratification or therapy response in diverse clinical settings.
Felix Meissner (UKB) felix.meissner@uni-bonn.de
Stimulation assay (DZNE)
The human immune system is most readily accessible through tissues and organs that do not require major surgical intervention, such as blood. In many instances, circulating immune cells reflect an individual’s health status and provide valuable insights into physiological and pathophysiological processes. Ex vivo stimulation of whole blood is a powerful tool for investigating immune responses. In the context of clinical research, applications of whole blood stimulation encompass host immunity, disease characterization, diagnosis, treatment evaluation, and drug development. At DZNE, ex vivo stimulation assays are performed utilizing TruCulture systems, which offer a robust and reproducible workflow for stimulating whole blood. This system is compatible with all downstream analyses described on this page.
Anna Aschenbrenner (DZNE) anna.aschenbrenner@dzne.de
Marc Beyer (DZNE) marc.beyer@dzne.de
Targeted Immunotyping
Isolation of specific cell subpopulation (DZNE, UKB, UBO)
While a systems immunology approach is crucial for understanding the immune system’s mechanisms and dynamics, in-depth analysis of specific cell populations or subpopulations remains vital for elucidating causality within molecular processes. At DZNE, we have established a diverse array of isolation methods, ranging from magnetic bead depletion or enrichment to fluorescence-activated cell sorting (FACS). The integration of high-dimensional cytometry with cell sorting capabilities, facilitated by the BD S8 cell sorter, enables the isolation of extremely rare cell types with high purity.
Marc Beyer (DZNE) marc.beyer@dzne.de
Lorenzo Bonaguro (DZNE) lorenzo.bonaguro@dzne.de
Natalio Garbi (UKB)
Elvira Mass (UBO)
TCR and BCR analysis (PRECISE)
B and T cell receptors (BCRs and TCRs) are crucial for adaptive immunity. Modern technologies allow for simultaneous analysis of B and T cell specificity, phenotype, and full-length receptor sequencing.
The PRECISE platform, collaborating with BD Biosciences, developed a groundbreaking multi-omics pipeline. This integrates transcriptomics, proteomics, and immune receptor sequencing at the single-cell level. For T cells, the addition of oligo-labeled dextramer technology (dCODE) enhances phenotyping by identifying antigen-specific T cells. This integrated approach provides a deeper understanding of immune responses and has significant implications for immunological research and the development of novel therapies.
Marc Beyer (DZNE) marc.beyer@dzne.de
Lorenzo Bonaguro (DZNE) lorenzo.bonaguro@dzne.de
Autoreactive Antibodies
Autoantibody analysis plays a pivotal role in immunotyping by providing crucial insights into the immune system’s function. These unique antibodies, erroneously directed against the body’s own tissues, serve as valuable markers for identifying and characterizing specific immune cell populations and underlying disorders. By analyzing the presence, types, and levels of autoantibodies, clinicians can gain valuable information for accurate diagnosis, assess disease activity and progression, and predict potential complications. This comprehensive understanding facilitates personalized treatment plans and improves overall patient outcomes in a variety of autoimmune conditions.
Cell type specific stimulation assays (DZNE)
Cell type-specific stimulation assays, performed after isolation of respective immune cell populations, offer a refined approach to investigating immune responses. These assays provide detailed insights into the behavior of specific immune cell types, allowing for more targeted analysis of physiological and pathophysiological processes. Isolation techniques, such as magnetic bead separation or flow cytometry sorting, enable the purification of distinct cell populations like T cells, B cells, or monocytes. Once isolated, these cells can be stimulated with specific activators, such as anti-CD3/CD28 for T cells or LPS for monocytes, to elicit cell type-specific responses. Cluster applicants have specialised in optimizing this approach for the standardized and high-throughput examination of direct effects of stimulants on particular cell types, without the complexity of whole blood interactions. These cell type-specific stimulation assays are valuable tools offering insights into cellular activation, cytokine production, and functional responses of individual immune cell subset.
Marc Beyer (DZNE) marc.beyer@dzne.de
Steering Committee
Prof. Dr. Joachim L. Schultze
PD Dr. Marc D. Beyer
Prof. Dr. Kerstin Ludwig
Prof. Dr. Felix Meissner
Dr. Anna Aschenbrenner
Dr. Lorenzo Bonaguro
Publications
- Kröger C, Müller S, Leidner J, Kröber T, Warnat-Herresthal S, Spintge JB, Zajac T, Neubauer A, Frolov A, Carraro C; DELCODE Study Group; Jessen F, Puccio S, Aschenbrenner AC, Schultze JL, Pecht T, Beyer MD, Bonaguro L. Unveiling the power of high-dimensional cytometry data with cyCONDOR. Nat Commun. 2024 Dec 19;15(1):10702. doi: 10.1038/s41467-024-55179-w.
- Leidner J, Theis H, Kraut M, Ragogna A, Beyer M, Schultze J, Schulte-Schrepping J, Carraro C, Bonaguro L. Cost-Efficient Transcriptomic-Based Drug Screening. J Vis Exp. 2024 Feb 23;(204). doi: 10.3791/65930.
- Carraro C, Bonaguro L, Srinivasa R, van Uelft M, Isakzai V, Schulte-Schrepping J, Gambhir P, Elmzzahi T, Montgomery JV, Hayer H, Li Y, Theis H, Kraut M, Mahbubani KT, Aschenbrenner AC, König I, Fava E, Fried HU, De Domenico E, Beyer M, Saglam A, Schultze JL. Chromatin accessibility profiling of targeted cell populations with laser capture microdissection coupled to ATAC-seq. Cell Rep Methods. 2023 Oct 23;3(10):100598. doi: 10.1016/j.crmeth.2023.100598.
- Bonaguro L, Schulte-Schrepping J, Ulas T, Aschenbrenner AC, Beyer M, Schultze JL. A guide to systems-level immunomics. Nat Immunol. 2022 Oct;23(10):1412-1423. doi: 10.1038/s41590-022-01309-9.
- Müller S, Kröger C, Schultze JL, Aschenbrenner AC. Whole blood stimulation as a tool for studying the human immune system. Eur J Immunol. 2024 Feb;54(2):e2350519. doi: 10.1002/eji.202350519.
- Verstegen NJM, Pollastro S, Unger PA, Marsman C, Elias G, Jorritsma T, Streutker M, Bassler K, Haendler K, Rispens T, Schultze JL, Ten Brinke A, Beyer M, van Ham SM. Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates. Elife. 2023 Mar 2;12:e83578.Meissner F, Geddes-McAlister J, Mann M, Bantscheff M. The emerging role of mass spectrometry-based proteomics in drug discovery. Nat Rev Drug Discov. 2022 Sep;21(9):637-654. doi: 10.1038/s41573-022-00409-3. Epub 2022 Mar 29.