News from … Part I

Department of Sports Medicine and Health Promotion, Friedrich-Schiller-University Jena, Jena, Germany
Department for Internal Medicine IV (Gastroenterology, Hepatology and Infectious Diseases), Jena University Hospital, Jena, Germany
Center for Sepsis Control and Care (CSCC), Jena University Hospital/Friedrich-Schiller-University Jena, Jena, Germany

What is the impact of acute anaerobic exercise on immune cells in individuals with and without B cell depletion in the context of post-viral infection syndrome? – A case study
Patients with post-acute infections syndromes (PAIS) suffer from a high degree of fatigue and exercise intolerance including post-exertional malaise (PEM). In healthy individuals and even cancer patients, physical exercise has been found to have a positive effect on health-related outcomes caused by immune cell modulation but little is known about the acute response to an exercise stimulus in PAIS patients. The aim of our study was to analyze immune cell modulation in PAIS patients performing a 1-minute anaerobic exercise test. Here, we present a special case of a young female athlete diagnosed with post-acute infection syndrome and treated with Rituximab for depleting B cells.

Our case study focused on a 20-year-old female patient with PAIS, who was treated with Rituximab for a chronic replicative Epstein-Barr virus infection. Peripheral blood was drawn on two time points: 6 months (T1) and 20 months (T2) after receiving the last dose of Rituximab, and additionally two sex/aged matched controls. Samples were collected before and after a 1-min sit-to-stand test at the following time points: base line (BL), post 5, 10, 15, 30, 45 min (P5, P10, P15, P30, P45). Three more sex/age-matched controls were added for the baseline data. Leukocyte subsets were profiled using MAXPAR DIPA assay and Cytometry by time of flight (CyTOF). Data analysis was manually performed using FlowJo v10.10.0.

Data from our PAIS patient show a sustained B cell depletion six months after the last dose of Rituximab (T1) and a plasma blast (PB) frequency higher than in the control group (N = 5). Both subsets were recovered to the normal level in the follow-up sample (T2). However, memory B cells were persistently decreased at T1 and T2. For the T cell subsets, the CD4/CD8 T cell ratio was lower at T1. In addition, persistent imbalance of Th1/Th2 ratio towards Th1 and persistent higher frequencies of CD8⁺ terminally differentiated effecter memory (TEMRA) T cells were observed at T1 and T2. In depth analysis revealed a special phenotype of CD8⁺ TEMRA cells: CD8⁺CCR7^–CD45RA⁺CD28^–CD57⁺CD56⁺CXCR3⁺ indicating an acquired natural killer (NK) cell trait. We found that the anaerobic exercise induced the following changes in the immune profile: I) a significant mobilization of absolute numbers of leukocytes immediately after exercise at P5 with a less slope of decline up to P45 in the patient samples compared to the control group (N = 2), II) an about 2-fold increase of NK cell frequencies referred to total CD45 at P5 followed by a reduction below the BL values in the patient and control samples, respectively, iii) the observed modulation was mostly prominent in the CD56⁺CD16⁺ NK cell subset – a phenomenon that has been described before in healthy individuals. Interestingly, we also observed a phenotype linked to myeloid derived suppressor cells (MDSC) exclusively after B cell normalization at P15, P30, P45, a phenotype that is associated to severely diseased COVID-19 patients.

This case study highlights the impact of anaerobic exercise on immune cell dynamics in a young female athlete diagnosed with PAIS and treated with Rituximab. The small stimulus induced changes in the immune cell profile including an immediate and transient mobilization of leukocytes and NK cells shortly after starting the exercise. The emerging of specific immune cells such as MDSC points to the complex nature of immune responses in PAIS patients and underscores the importance for further research to verify these observations.

Charité – Universitätsmedizin Berlin

Putting TeMals to work: Multiplexing of Matrigel embedded, patient-derived organoids using thiol-reactive barcodes
Multiplexing in mass cytometry eliminates the confounding effects of technical variabilities in staining, processing, and data collection when comparing multiple samples. The most common multiplexing workflow is based on commercially available, amino-reactive palladium based barcodes. While this method works very well for a multitude of fixed cell types, it poses some challenges for other applications such as 3D cell culture models. These culture models involve cells that are embedded in protein rich, basement membrane like gels (e.g. Matrigel) in low cell numbers. Therefore, a barcoding workflow that enables sample pooling at the earliest possible stage and possibly allows for in situ-barcoding is desirable. A new kind of thiol-reactive, tellurium based barcodes (TeMals) has been introduced and proven suitable for 3D organoid culture. Here, we present our implementation of a TeMal based barcoding approach for patient-derived colorectal cancer organoids, including downstream debarcoding and sample analysis.

Department of Environmental Immunology, Helmholtz Centre for Environmental Research – UFZ and Leipzig CyTOF Team at UFZ, Leipzig, Germany

Applications of CyTOF-based immunophenotyping: a case example of chemical mixture effects
Introduction: Modern populations are daily exposed to complex chemical mixtures. Per- and polyfluoroalkyl substances (PFAS) are of public health concern because they bioaccumulate and adversely affect the immune system. Nonetheless, most studies have addressed PFOS and PFOA immunotoxicity, while neglecting the potential impact of other PFAS and their mixtures. Here, we assess whether a mix of PFAS at concentrations detected in European populations modifies the immunophenotyping of human peripheral blood mononuclear cells (PBMCs) by using CyTOF.

Methods: PBMCs were isolated from 6 healthy donors and cultured with a PFAS mixture at 1x, 100x, and 1000x the human blood concentration for 24 h. The mixture was dissolved in DMSO and contained 6 different PFAS (PFOS, PFOA, PFHxS, PFNA, PFDA, and PFUnDA) with 1x concentrations ranging from 0.19 to 22.34 ng/mL. After exposure, 1.0×10⁶ cells/mL were harvested, stained with the Maxpar Direct Immune Profiling Panel, and acquired in a Helios System.

Results: tSNE high-dimensional reduction and clustering analysis revealed that PFAS-1000x induced marked changes in the expression of cell surface markers among 33 immune subpopulations without affecting their viability. The most susceptible subsets were B and T lymphocytes. Manual gating showed a concentration-dependent decrease in the naïve/memory B cell ratio. Likewise, the ratio of central/effector memory CD4⁺ T cells exposed to PFAS-100x was significantly higher as compared to the DMSO control.

Conclusions: This preliminary data indicates that the tested PFAS mixture alters the immunophenotyping of PBMCs following acute exposure in vitro. Further experiments with greater sample sizes and functional assays are ongoing.

Core Facility Cytometry, Medical Faculty, Ulm University, Ulm, Germany

Start with CyTOF: Step by step to success from the perspective of a core facility

Starting a CyTOF study is exciting but sometimes also challenging.  We are presenting an example of the work flow from our core point of view: from the first discussion, to the first tests, towards the actual samples acquiring and workflow documentation. We will also touch base about some issues we encountered in the hope that other core facilities will benefit from our experience.

University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne

Expanding high-dimensional analyses of aggressive lymphoma, the tumor microenvironment, and extracellular vesicles

In Cologne, the CRC1530 focuses on elucidating and targeting the pathogenic mechanisms that underlie B cell lymphoma, emphasizing understanding how malignant B cells and their local tumor microenvironment interact. Within this framework, our CyTOF and imaging mass cytometry (IMC) laboratory enable comprehensive analyses of tumor cells and their microenvironmental partners in mantle cell lymphoma, chronic lymphocytic leukemia (CLL), and CAR T cell-treated diffuse large B cell lymphoma. Notably, we recently supported a study revealing that LYN-kinase in leukemic cells can program stromal fibroblasts to promote leukemic survival (Vom Stein et al., Nat Commun., 2023) underscoring the critical role of high-dimensional analyses of the tumor microenvironment in B cell malignancies.

Despite these advances, numerous mechanisms of aberrant cellular communication remain poorly understood, particularly those involving extracellular vesicles (EVs). EV-mediated intercellular communication is especially challenging, as traditional bulk analyses often provide only low-dimensional insight into EV composition and their recipient-cell profiles. Consequently, the field urgently needs approaches leveraging single-cell resolution to capture the heterogeneity of EV uptake and function in life sciences research.

To address these gaps, we aim to develop novel MC- and IMC-based methodologies that afford high-dimensional single-cell analyses of EV-recipient cells, focusing on CLL. In this regard, we have developed a tellurium-based metabolic mass-tag labeling strategy that marks the proteome of secreted EVs. By applying TeLEV in various B cell disease models and validating results in patient-derived samples, we can visualize EV uptake, characterize their molecular signatures in recipient immune cells, and assess their biological impact with unprecedented resolution.

German Rheumatology Research Center Berlin – A Leibniz Institute, Berlin, Germany

A Mass Cytometry Workflow for Ex-Vivo Phosphorylation and Mucosal Immune Profiling in Systemic Lupus Erythematosusbstract

Systemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disease characterized by immune tolerance breakdown and the production of IgG autoantibodies. Even though the mechanisms of disease progression, clinical manifestation and therapy response in SLE are highly heterogeneous and poorly understood, recent evidence suggests an involvement of the gut-immune axis in both SLE pathogenesis and prognosis. This highlights the need to investigate mucosa-related immunity as a potential source of patient heterogeneity.

To explore the role of mucosa-related immunity in SLE, we established a robust mass cytometry workflow for in-depth ex-vivo phosphorylation profiling and comprehensive mucosal immune phenotyping of peripheral blood. The workflow is optimized for the analysis of fixed whole blood samples, scalable to large clinical studies, and specifically designed to maximize data output from limited sample material.

The sample processing pipeline uses a tailored cell sorting strategy to divide whole blood into lymphoid and myeloid fractions, which are subsequently analyzed with two optimized >50-plex antibody panels. These panels include markers that identify key mucosal-associated immune populations, such as IgA+ plasmablasts, γδ T cells, MAIT cells, invariant NKT cells, and other leukocytes with gut-homing properties, expressing, e.g. CCR9, CD69, and the integrins α4β7 or αEβ7. Additionally, the panels incorporate phosphoprotein-specific antibodies to measure ex-vivo activity of multiple intracellular signaling pathways.

Overall, this workflows allows for deep phenotyping of peripheral blood, with focus on gut-related immune populations, and the analysis of intracellular phospho-signaling activity by mass cytometry. By providing a comprehensive view of mucosal immunity, this approach may shed light into role of the gut-immune axis in SLE and identify potential sources of disease heterogeneity that could be exploited for tailored clinical management.

Department of Medicine II (Gastroenterology, Hepatology, Endocrinology and Infectious Diseases), Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Abstract

Immunotherapies using checkpoint blockade antibodies are now utilized in Hepatocellular carcinoma (HCC) patients but prediction of responding patients remains difficult.  We utilize mass cytometry to profile the dynamic changes of the peripheral immune response to immunotherapy and utilize imaging mass cytometry to dissect the immune architecture in the tumor microenvironment.

I will discuss data from translational work that identifies key responding immune populations in patients exposed to checkpoint blockade in HCC. Our data indicates significant remodeling of the peripheral immune compartment involving distinct CD4 and CD8 T cell subsets. The response dynamics may help predict therapeutic outcomes and could support the development of more personalized treatment strategies for HCC.