Poster Session

Technical University of Munich (TUM), School of Medicine, Institute for Medical Microbiology, Immunology and Hygiene, Munich 81675, Germany

In vitro induction of exhaustion in human CD8+ T cells via mitochondrial inhibition and ectopic TOX expression
In vitro manipulation of human exhausted T cells (Tex) is challenging due to the low numbers of Tex that can be isolated in human disease and absence of a consensus model to induce exhaustion. To overcome this and to recapitulate human Tex development in vitro, we generated a model that integrates major cues driving T cell exhaustion, such as TCR signaling, mitochondrial dysfunction induced by pharmacologic mitochondrial translation inhibition and TOX overexpression. Using CyTOF profiling, flow analysis and RNAseq analysis we identified that metabolic dysfunctionality enhances the differentiation to terminally exhausted CD127^–PD-1^hi cells with reduced cytokine production and gene expression programs, but also induces a significant fraction of exhausted T cell precursor populations (Tpex). Ectopic TOX expression further drives exhaustion programs with enhanced inhibitory receptor expression and reduced cytokine release. These models provide opportunities to study distinct stages of Tex development. Transcriptional analysis linked FOXO1 gene signatures to the enrichment of Tpex cells in the mitochondrial inhibition model. CRISPR knockout of FOXO1 led to a collapse of Tpex and severe Tex populations, validating the in vitro model as suitable to dissect the transcriptional regulators of T cell exhaustion.

Department of Internal Medicine II, Medical Center – University of Freiburg, Freiburg, Germany

Recent advances in establishing RNA detection by IMC
Classical IMC is confined to detection of protein markers using metal-labeled antibodies. Recently the range of Hyperion-detectable macromolecules has been expanded to RNA by combining advanced in situ hybridization techniques with metal-labeled oligonucleotide probes. This novel technique promises sequence-specific and sensitive detection of cellular, viral or bacterial RNAs with high spatial resolution, instrumental for e.g. identification of chemokine producing cells or virus replicating cells. Additional antibody staining enables co-detection of RNA and protein.

We have started to establish Hepatitis B Virus (HBV) RNA detection in our lab employing the „RNAscope HiPlex-IMC“ system commercially available from Advanced Cell Diagnostics. RNAscope, primarily developed for fluorescent RNA detection, can be adapted to IMC by user performed conjugation of metals to sulfhydryl-modified oligonucleotides using the Standard Biotools’ MaxPar Kit.

To test the performance of the assay we used a hepatoma cell line stably transfected in vitro with HBV plus untransfected cells as negative control. Tissue-like FFPE samples were generated by formalin fixation and paraffine embedding of cultured cells. FFPE sections were stained for HBV RNA by sequential hybridization with (i) a HBV specific (or neg. control) probe, (ii) signal amplifier probes and (iii) an orthogonal metal-labeled imaging probe. Images aquired with the Hyperion system revealed HBV probe dependent signal accumulation exclusively in cells from HBV positive samples strongly arguing for specific detection of HBV RNA. Our data demonstrate proof-of-principle for IMC-mediated sequence-specific RNA detection and provide a basis for future implementation of RNAscope into our IMC analysis pipeline.

Deutsches Rheuma-Forschungszentrum Berlin, a Leibniz Institute, Berlin, Germany

SplitSOM automates debarcoding of mass cytometry data
Sample barcoding offers the advantage of measuring multiple samples from different sources simultaneously, thereby effectively harmonizing assay preparation and sample acquisition conditions. Debarcoding, i.e. the extraction of single sample data from barcoded data convolutes is a crucial step in mass cytometry data curation workflows. Traditionally, debarcoding is performed by manual gating, although algorithmic solutions have also been developed for this task. However, such solutions showed suboptimal performance on our surface barcoding on live cells.

Here, we introduce SplitSOM, a novel computational debarcoding method utilizing FlowSOM, a widely accepted clustering method for cytometry data.

In contrast to existing debarcoders, SplitSOM considers the entire information of cellular neighborhood based on overall barcode stainings to group cells with similar barcode signatures, rather than assessing and assigning each cell individually. Additionally, SplitSOM can automatically resolve different barcode configurations, including examples using varying numbers of barcode markers.

We evaluated the recovery and assignment accuracy of SplitSOM. In a 20-sample dataset with a 6-choose-3 barcode configuration, the events-to-sample assignment rate was 92%, closely matching the results of manual gating. We further investigated the rate of cell misclassification after successful SplitSOM debarcoding by analyzing a secondary (CD45) barcode not used for debarcoding. We assessed the cross-contamination between three samples, each stained with a different CD45 barcode. The cross-contamination was less than 0.17%, indicating minimal contamination between the debarcoded samples.

In summary, our newly developed SplitSOM debarcoder permits quick, flexible, accurate and efficient debarcoding of mass cytometry data, facilitating swift and automated data curation in high throughput facilities.

Department of Internal Medicine II, Universitätsklinik Freiburg, Universität Freiburg, 79106 Freiburg, Germany

Imaging Mass Cytometry Protocol for Formalin-Fixed Paraffin-Embedded Mouse Tissues: Challenges and Opportunities in Analyzing Archival Specimens
Imaging mass cytometry (IMC) has recently become a widespread method for spatial analysis of immune and stromal components in a range of diseases, including solid tumors. The majority of studies have focused on patient samples. In contrast, the application of IMC to animal tissues remains limited, including the gastrointestinal tract of C57BL/6 mice.

Here we established a highly multiplexed IMC panel to apply IMC on formalin-fixed paraffin-embedded (FFPE) gastrointestinal mouse tissues, with the goal to specifically interrogate stomachs of the B6.IL-1beta^(EBV)tcw mouse model, which is used to study gastroesophageal adenocarcinoma development. To address challenges associated with older FFPE-blocks, our approach encompasses specimen collection, fixation, antibody validation via immunofluorescence, metal tag conjugation, and IMC validation, as well as validation for IMC analysis. The intricacies of working with FFPE tissues include insights into specimen processing, antigen retrieval, and blocking steps to enhance data quality. We tested over 70 antibodies using the Hyperion® imaging system with a 63% success rate of obtaining IMC signal after in-house conjugation. Our designed signature panel allows for spatial analysis of distinct cell types (mesenchymal, stromal, immune, epithelial), and processes involved in cancer development including epithelial-to-mesenchymal transition, a wide range of immune cell processes (e.g. polarization and cytotoxicity), signaling pathways, as well as the overall tissue architecture (E-Cadherin, Fibronectin, Collagen and β-tubulin 3).

Our work will serve as a resource to explore immunological and stromal landscapes of existing murine FFPE specimens using highly multiplexed imaging. This protocol allows for analyzing older FFPE blocks with IMC technology, leveraging these resources to accelerate scientific discovery.

DRFZ, ein Leibniz-Institut, Berlin

NFDI4Immuno: Building a framework for comprehensive immunological data integration and analysis, collaboration and Open Science

In the era of advanced single-cell technologies, particularly mass cytometry, capable of generating very information-rich datasets, the necessity for data sharing and reuse has grown significantly in immunological research. To address this need, the National Research Data Infrastructure for Immunology (NFDI4Immuno) initiative has been founded.

Here, we present how the NFDI4Immuno initiative aims to integrate immunological data and metadata from various experimental technologies, including cytometry, sequencing, immunoassays, and imaging, to provide a holistic view of immunological processes. The initiative strives to enhance scientific collaboration by harmonizing data representations, metadata standards, ontologies, and programmatic interfaces with other NFDI consortia, promoting seamless queries and cross-referencing. The project is committed to support users in effectively utilizing its resources and to foster the adoption of FAIR principles (findability, accessibility, interoperability and reusability) within the immunological community to contribute to the broader cultural shift towards Open Science. Finally, NFDI4Immuno plans to establish and manage a network of federated repositories for immunological data, to develop tools and services that facilitate standardized and reproducible data analyses, reinforcing scientific rigor and transparency.

Deutsches Rheuma-Forschungszentrum Berlin, a Leibniz Institute, Berlin, Germany

Antibody Isotype and Subclass Expression Shapes Plasma Cell Heterogeneity in the Human Bone Marrow

Mature plasma cells in the human bone marrow (BMPCs) are essential for humoral immunity and memory and may contribute to autoimmunity through the secretion of autoantibodies. Significant phenotypic heterogeneity among BMPCs has been reported, including the loss of traditional B cell markers like CD19 and the expression of atypical receptors such as CCR2, CD56, and CD28. However, the drivers and implications of this heterogeneity remain poorly understood.

We conducted an in-depth characterization of human bone marrow plasma cells (BMPC) of 9 donors using a 48-marker mass cytometry assay resolving all human Ig isotypes and subclasses, and targeted single-cell sequencing from 4 donors, including a 62-marker AbSeq panel, quantification of 399 mRNAs, and BCR sequence information, together enabling detailed investigation of BMPC heterogeneity in relation to Ig isotype expression.  IgG (48%) was the most abundant isotype among BMPCs, followed by IgA (41.5%) and IgM (3.2%). Within the IgG+ BMPC population, IgG1 and IgG2 were predominant, while IgG3 and IgG4 were detected at lower frequencies. Notably, IgG1+ and IgM+ BMPCs were enriched in CD19^neg BMPC, a phenotype previously linked to PC longevity. Additionally, IgG1+ BMPCs were enriched in a novel CD19-CD56+ subset, whereas IgA1+ BMPCs were more common among CD19+CD45- BMPCs. Ongoing analyses aim to systematically map the relationship between BMPC phenotype and Ig subclass, linking these characteristics to IgVH properties to elucidate clonal relationships and levels of somatic hypermutation across BMPC phenotypes. Both assays provided consistent information, underlining the validity of the results obtained by mass and genomic cytometry.

Our findings reveal that BMPC phenotypes are strongly associated with Ig isotype and subclass expression. This suggests that BMPC phenotypes and potentially their functions are pre-determined during class-switch recombination (CSR) in germinal centers Thus, CSR may not only have a critical role in defining antibody function but also in shaping the phenotypes of BMPC, which may regulate PC localization and persistence of individual PC in the bone marrow.

Department of Clinical Medicine, University of Bergen, Bergen, Norway and Department of Neurology, Neuro-SysMed, Haukeland University Hospital, Bergen, Norway

CNS Immune Profiling and Microglial Dynamics in Progressive MS Using Mass Cytometry

Multiple Sclerosis (MS) is a chronic inflammatory condition of the central nervous system (CNS) characterized by myelin loss and axonal damage. Most patients initially experience relapsing-remitting MS (RRMS), involving episodes of immune cell infiltration and inflammation in demyelinating lesions, followed by remission. Progressive forms, including primary progressive MS (PPMS) and secondary progressive MS (SPMS), lead to gradual disability without relapses, driven by CNS-restricted inflammation. Understanding the role of CNS-resident microglial cells in these processes is critical in progressive MS.

This study employs imaging mass cytometry (IMC) to analyze immune cells in cerebrospinal fluid (CSF) and post-mortem CNS tissues from progressive MS patients. An extensive microglial phenotyping panel, including TMEM119, MerTK, P2Y6, Iba1, and CD68, was developed to profile microglial activation states in white matter smouldering lesions and iron rims. Validation included microglial cell lines, iPSC-derived microglia, PBMCs, tonsils, healthy brain tissue, and MS-affected brain tissue.

Preliminary results show the panel distinguishes resting microglia, activated microglia, and infiltrating macrophages. Activated microglia exhibit a pro-inflammatory phenotype, enhancing understanding of CNS inflammation in progressive MS and RRMS patients with smouldering lesions.

no publishing

Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany

How to use iodinated Hoechst reagent in suspension mass cytometry?

Cell identification in mass cytometry relies on their sufficient labeling with a metal-labeled probe, which is routinely achieved by post-permeabilization staining with an iridium-labeled DNA intercalator. The detection of doublets is usually achieved by using the DNA content and event length but this practice is not sufficient. Cell membrane permeant Hoechst dyes are commonly used in flow cytometry to detect selectively live nucleated cells. By modifying Hoechst dyes with a metal isotope it can be used for mass cytometry approaches as well.

Here we analyzed whole blood samples stained with commercially available Maxpar DIPA-kit including ¹²⁷I-Hoechst dye only as well as in combination with iridium intercalator in order to compare the performance to samples stained exclusively with iridium intercalator.

Preliminary data show a rather limited discrimination of doublet cells when incubating ¹²⁷I-Hoechst with antibodies. However, this shortened protocol allows to circumvent the permeabilization step in sample preparation. Results show similar frequencies to iridium-stained samples for all major leukocyte subsets. When ¹²⁷I-Hoechst is utilized in addition to iridium intercalator the differentiation of doublets from single cells is more effective. As a result we suggest ¹²⁷I-Hoechst only staining for applications requiring same-day analysis such as antibody titrations and panel testing. For use cases such as clinical studies in which a better resolution between doublets and singlets is desired, an adoption of ¹²⁷I-Hoechst staining into standard iridium-based protocol is recommended. Altogether, we propose to include ¹²⁷I-Hoechst into mass cytometry assays.

Department of Internal Medicine II, University Hospital Freiburg, Germany

CD161 expressing CD4+CXCR3+ T cells accumulate in the liver of obese patients with MASH

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is emerging as a leading cause of chronic liver disease in western countries. Hepatic steatosis can progress to inflammation (Metabolic Dysfunction-associated Steatohepatitis or MASH), with a suspected role of hepatic T cells driving this process.

Methods: The study involved 51 obese patients undergoing bariatric surgery, stratified into groups with few (n=33) and high (n=18) inflammation levels according to histologic scoring and clinical parameters. Liver and blood samples were characterized by flow cytometry, validated by mass cytometry and subsequently further investigated by single cell RNA sequencing for a broad range of immune cell subsets.

Results: CD4 T cells were significantly more abundant in inflamed liver tissue. Cluster analysis identified a distinct CD161-expressing CD4+CXCR3+ T cell subset enriched in high-inflammation patients. In contrast, CD8 T cells and other immune cells showed no significant differences between the groups. Mass cytometry revealed additional defining markers: CD26, PD-1, CD127, and Tbet. Functional analysis showed increased pro-inflammatory cytokines and cytotoxic granules (Granzyme B and IL-17A) in liver and blood. These inflammatory changes were dynamic, subsiding over 12 months post-weight loss. Furthermore, significant positive correlations could be observed between the specific inflammatory T cell subsets and clinical and histological parameters indicative of liver damage.

Conclusions: Our findings reveal critical immunological mechanisms in obesity-related liver inflammation. The CD161-expressing CD4+CXCR3+ T cell population emerges as a potential diagnostic and therapeutic target for MASLD. The inflammatory changes‘ reduction after weight loss suggests targeted interventions could modulate disease progression, offering promising insights into modulating metabolic liver disease.

Standard BioTools

Whole Slide Imaging Modes and Curated Antibody Panels for Imaging Mass Cytometry Approach Reveal Extensive Spatial Heterogeneity of Human Glioblastomas

Glioblastoma (GBM) presents a complex form of brain cancer that is challenging to diagnose and treat. Gaining spatial insights into the cellular composition of GBM tissue has tremendous potential to inform clinicians and researchers about mechanisms behind spatial predictors of treatment success and disease etiology and progression.

Imaging Mass Cytometry™ (IMC™) is a high-plex spatial biology imaging technique that enables deep characterization of the diversity and complexity of GBM and other tumor microenvironments (TMEs). IMC supports detailed assessment of cell phenotype and function using 40-plus metal-tagged antibodies simultaneously on a single slide without artifacts associated with fluorescence-based spectral overlap, tissue autofluorescence, multiple acquisition cycles and tissue degradation.

Specifically designed for high-throughput applications and whole slide imaging (WSI) modes, the Hyperion XTi™ Imaging System with 40-slide loader permits automated and continuous imaging of more than 40 large tissue samples (400 mm2 per tissue) per week. We showcase the application of WSI using curated antibody panels to study the complexity of the GBM TME.

Department of Dermatology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

Cross talk between skin and joints impairs bone homeostasis in psoriatic arthritis

Psoriasis and psoriatic arthritis (PsA) are chronic inflammatory disorders with interconnected pathologies involving the skin and joints. Innate lymphoid cells (ILCs) have emerged as key players in the transition from skin inflammation to joint destruction, yet the mechanisms underlying this „skin-joint axis“ remain incompletely understood. This study leverages Imaging Mass Cytometry (IMC) to investigate the spatial distribution and chemokine receptor expression of ILCs in lesional and non-lesional skin from psoriasis patients with and without PsA.

Our previous flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) from psoriasis patients and healthy controls revealed a significant increase in ILCs in patients, with elevated expression of the chemokine receptor CXCR3 in ILC2s, a receptor linked to immune cell recruitment to inflamed tissues. Extending these findings to the psoriasis tissue microenvironment using IMC, we observed high CXCR3 expression in ILCs identified within the lesional skin. Additionally, CXCL16, a skin-homing chemokine, was predominantly expressed in the epidermis and in monocytes. These observations suggest key roles for CXCR3 and CXCL16 in mediating ILC migration and tissue infiltration. Future comparative analyses with PsA lesional tissue will focus on these chemokines to better understand the migratory nature of ILCs from skin to joints.

This spatially resolved approach provides comprehensive immune profiling of psoriatic skin, comparing lesional and non-lesional sites with healthy controls to identify early disease-driving factors. By linking IMC data to clinical PsA status, we aim to uncover biomarkers predictive of disease progression and elucidate mechanisms facilitating the migration of skin-derived immune cells to joint tissues.

Translational Systems Immunology, Hepatogastroenterology, Clininc for Internal Medicine II, University Hospital Freiburg

First impression of the new transparent polymer-coated slides for use in multiplex ion beam imaging (MIBI) mass cytometry

Introduction: The MIBIscope is a recent addition to the field of tissue imaging by mass spectrometry using time-of-flight secondary ion beam imaging for simultaneous detection of 40+ metal-labeled antibodies at subcellular resolution down to 0.39 µm per pixel. The MIBIscope works with a 30keV-activated primary xenon ion beam, which is grounded by a conductive gold coating on the slide. However, the opaque gold layer does not allow traditional light-based approaches in addition to standard MIBIscope imaging. To overcome this issue, slides with a conductive but transparent blue top layer have been developed. We have tested the performance of two pre-production blue slides and compared them with the gold standard.

Methods: Four µm thick sections of a tissue microarray (TMA) were cut, mounted and stained in parallel using a single antibody mastermix. After individual background removal, blue and gold data were segmented and analysed in parallel.

Results: Blue slides showed a trend for better tissue adhesion. Blue slides showed slightly less background noise and slightly higher signal intensity before and after background removal. Consequently, segmentation and analysis of blue slide data resulted in a slightly lower number of undefined cells in unsupervised clustering and subsequently better segregation between immune cells and non-immune cells.

Conclusion: Compared to gold slides, blue slides offer transparency and, at first impression, better tissue adhesion, slightly less background noise and slightly improved signal intensity. Segmentation and clustering are improved accordingly.

Division/Center of Pediatric Hematology/Oncology at Children’s Hospital of Zhejiang University School of Medicine, China

Mass Cytometry in Pediatric Hematological Research and Potential Clinical Applications

Substantial progress of high content cytometric detection has been made in pediatric hematology-oncology diagnosis in recent years. Defining complex antigenic profiles that are associated with specific cytogenetic/molecular defects, as well as systemic immunomonitoring for programmed chemo-immuno treatment, has significantly contributed to more accurate prognosis and improved efficacy. Mass cytometry holds a promise for being a more standardized and comprehensive cytometric detection technology. In this talk, we would like to discuss it’s current and future applications in hematological research and diagnosis, combined with novel bioinformatic toolbox and immunotherapeutic interventions.

Department of Pathobiology (Microbiology), University of Veterinary and Animal Sciences, Lahore (Narowal Campus) Narowal 51600, Pakistan

Silver Nanoparticles Coated AZ31: A Nanotech Defender in the War Against Foot-and-Mouth Disease Virus

Background:

            The economically important, foot-and-mouth disease (FMD) is extremely contagious disease of cloven-hoofed animals caused foot-and-mouth disease virus (FMDV). There isn’t an antiviral treatment available for FMDV right now.  The bactericidal effect of Ag NPs/PEI/MAO (APM) coated AZ31 is well established. The purpose of this work is to evaluate the APM coated AZ31’s in vitro antiviral activity against FMDV.

 Methodology:

            Using the Baby Hamster Kidney (BHK-21) cell line cultured in GMEM medium supplemented with 10% Fetal Bovine Serum, the cytotoxicity and antiviral characteristics of APM coated AZ31 will be observed. APM coated AZ31 will be prepared according to Xin Wang et al., 2021. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide test, the viability of the cells will be assessed.  The culture medium was removed, and ten-fold serial dilutions of FMDV in a serum-free medium were added and then incubated for one hour at 37°C. The plaque reduction assay will be used to assess the direct effects of the FMDV strain O/ME-SA/PanAsia-2/ANT-10 on APM coated AZ31 in both the extracellular (virucidal assay) and different stages of virus replication (antiviral assay) phases. The Nano-Particles (NPs) will be introduced to the cells at the pre-attachment, attachment, and post-penetration stages of the viral infection cycle to ascertain the mode of their antiviral activity. The NPs will be dissolved in distilled water and utilized at doses of 25, 50, 100, 150, 200, and 250 μg/ml to evaluate the antiviral activity.

Results:

Anticipated outcomes indicate that APM-coated AZ31 will be safe in BHK-21 cell line at concentrations up to 250 μg/ml. The NP treatments suggested that the APM coated AZ31 would have antiviral and virucidal effects in vitro. APM coated AZ31 can inhibit FMDV by more than 90% during the early phases of infection, such as attachment and penetration, but not after penetration, according to the plaque reduction assay.

Conclusion:

The inference made is that APM coated AZ31 may be administered locally as an antiviral medication to susceptible animals in the early stages of infection.

Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany

High throughput spatial immune mapping reveals an innate immune scar in post-COVID-19 brains

The underlying pathogenesis of neurological sequelae in post COVID-19 patients remains unclear. Here, we used multidimensional spatial immune phenotyping methods on brains from initial COVID-19 survivors to identify the biological correlate associated with previous SARS-CoV-2 challenge. Compared to healthy controls, individuals with post COVID-19 revealed a high percentage of TMEM119⁺P2RY12⁺CD68⁺Iba1⁺HLA-DR⁺CD11c⁺SCAMP2⁺ microglia assembled in prototypical cellular nodules. In contrast to acute SARS-CoV-2 cases, the frequency of CD8⁺ parenchymal T cells was reduced, suggesting an immune shift towards innate immune activation that may contribute to neurological alterations in post COVID-19 patients.

Department of Medicine II, University Hospital Freiburg – Faculty of Medicine, University of Freiburg, Freiburg, Germany and Faculty for Biology, University of Freiburg, Freiburg, Germany

ISO: Image analysis of Subcellular localization and spatial Overlap in tissue cells
Generalist segmentation algorithms like Cellpose [Pachitariu, M., & Stringer, C. et al. (2022)] and DeepCell Mesmer [Greenwald, N. F. et al. (2022)] enabled fast and reliable tissue segmentation. Yet these segmentation algorithms do not fully cover analysis of subcellular localization of proteins, since they only take into account segmentation masks of the full cell and the nucleus. Here, we developed an algorithm for the detailed analysis of subcellular localization based on image coordinate information and a python based analysis pipeline to accommodate this algorithm.
As application examples, we analyzed the nuclear translocation of the cAMP dependent transcription factor 6 (ATF6) and nuclear versus cytoplasmic localization of the focal adhesion kinase (FAK) in hepatocellular carcinoma. Where we could show improved characterization.
Another benefit of this approach was the increased sensitivity in transcription factor analysis of immune cells, e.g., FoxP3 in Tregs. By this a more comprehensive and detailed profiling of the cellular landscape and a better understanding of the cellular state of immune cells in the tissue was achieved. Hence, the ISO algorithm provides a versatile tool for imaging analysis not only enabling determining subcellular localization of proteins but also to increase the detection sensitivity.