Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Biomedical Center Faculty of Medicine Charles University Pilsen Czech Republic

Charles Perkins Centre The University of Sydney Sydney New South Wales Australia

Department of Microbiology Faculty of Medicine University Hospital in Pilsen Charles University Pilsen Czech Republic

Discipline of Child and Adolescent Health The University of Sydney Sydney New South Wales Australia

Human Cancer and Viral Immunology Laboratory The University of Sydney Sydney New South Wales Australia

Kids Research The Children's Hospital at Westmead Sydney New South Wales Australia

Liver Injury and Cancer Program Centenary Institute Sydney New South Wales Australia

Nepean Clinical School The University of Sydney Sydney New South Wales Australia

Nepean Hospital Nepean Blue Mountains Local Health District Penrith New South Wales Australia

Ramaciotti Facility for Human System Biology The University of Sydney and Centenary Institute Sydney New South Wales Australia

School of Medical Sciences Faculty of Medicine and Health The University of Sydney Sydney New South Wales Australia

Sydney Cytometry Core Research Facility Charles Perkins Centre The University of Sydney and Centenary Institute Sydney New South Wales Australia

Sydney Nano The University of Sydney Sydney New South Wales Australia

The University of Sydney Institute for Infectious Diseases The University of Sydney Sydney New South Wales Australia

Viral immunopathology Laboratory Infection Immunity and Inflammation Research Theme School of Medical Sciences Faculty of Medicine and Health The University of Sydney Sydney New South Wales Australia

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