Poster #32 - Kelsey Keith

Broad immunophenotyping panel with supervised immune cell classification reveals age-dependent differences between mitochondrial disease subjects and healthy controls

Matei Ionita1,2, Richard Schretzenmair1, Derek Jones1, Kelsey Keith3#, Matthew Sullenberger4, Benjamin R. Fischer4, Hakon Hakonarson3,5, Jonni Moore1, Marni J. Falk4,5* 1Penn Cytomics & Cell Sorting Shared Resource Laboratory, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 2Institute for Immunology and Immune Health, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 3Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia, Philadelphia, PA 4Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 5Center for Applied Genomics, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 6Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA *Corresponding author #Presenting author

Primary mitochondrial diseases (PMDs) are a highly heterogenous group of genetic disorders, with multi-system symptoms ranging in severity and timing of clinical progression. Immune system dysfunction is common in PMD, where patients are often vulnerable to disease progression at the time of viral infections and may not completely respond to vaccination. Understanding of this dysfunction in PMD patients has been limited, with existing work largely limited to case reports or examination of a single cell type in a single PMD disorder. To systematically characterize the immune system in diverse PMD patients, deep cell immunophenotyping of peripheral blood mononuclear cells (PBMCs) was performed on a spectrally enhanced BD Symphony X50 instrument using a customized 41 parameter flow cytometry panel designed for simultaneous broad typing of lymphocytes and monocytes. Analysis was performed with an automated computational pipeline, classifying cell types in a hierarchical fashion to assign each cell as debris or to one of 8 major cell types. Major cell types were then divided into predetermined phenotypes ending with 56 immune sub-features, represented as proportions of the major cell populations. Testing for association between PMD and immune features was performed separately in pediatric subjects, defined as less than 18 years old, and adult subjects. In the pediatric cohort, 19 of 56 features were significantly different, and were associated with more mature/exhausted T cells. No significant differences were found in PMD adults. Principal component analysis (PCA) also revealed an immune maturity axis, progressing from pediatric controls to pediatric cases, then to all adults irrespective of disease status. This study delineates a unique immune signature in pediatric PMD, with heightened immune cell maturity and senescence, which may contribute to immune dysregulation in affected individuals. Furthermore, the novel 41-parameter immunophenotyping panel and computational methodology established in this work offer broad applicability to human immunology research.