Integration and analysis of high-dimensional single-cell cytometry and imaging data in a murine model of viral encephalitis


Emerging infectious diseases such as viral encephalitis drive a complex immune response where the inflammatory process intended to eliminate an invading pathogen contributes to significant immunopathology, both at the site of infection and more systemically. To understand this response, careful analysis using high-dimensional (HD) cytometry, imaging, and single-cell technologies are required. As the size and complexity of HD data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. Moreover, approaches that allow for the integration of data across batches, experiments, and technologies are not well incorporated into computational toolkits to allow for streamlined workflows. To address these challenges, we expanded our analysis toolkit ‘Spectre’ to facilitate the analysis of any form of high-dimensional single-cell or imaging data, incorporating extensive functionality to integrate data across batches, experiments, or technologies. Additionally, we developed novel boundary-based cell-segmentation approaches using Ilastik, dramatically improving the accuracy and stringency of segmentation in complex heterogenous tissues. Here we use Spectre to comprehensively map cellular infiltration into the central nervous system in a mouse model of viral encephalitis, incorporating both HD flow cytometry and Imaging Mass Cytometry data, revealing key response patterns associated with disease severity and progression over time.


Dr. Thomas Ashhurst is a high-dimensional cytometry and single-cell specialist with the Sydney Cytometry Core Research Facility, and an Honorary Research Fellow with the Infection, Immunity, and Inflammation Theme at The University of Sydney. In these roles he leads the development and application of a number of high-dimensional cytometry, imaging, and single-cell sequencing technologies, providing advanced training and consultation for researchers. He also leads a multi-disciplinary ‘systems immunology’ initiative, seeking to comprehensively map the immune response to inflammation and emerging infectious diseases, including viral encephalitis and COVID-19, using high-dimensional single-cell and imaging methods in addition to advanced computational analysis approaches.


High-Dimensional Cytometry Specialist
Sydney Cytometry Core Research Facility

Honorary Research Fellow
Infection, Immunity, and Inflammation Theme
School of Medical Sciences
Faculty of Medicine and Health

The University of Sydney