News from … Part I

Abstract

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.

Affiliation

Abstract

Affiliation

Abstract

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

AMass 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.