Development of a Spectral Flow Cytometry Analysis Pipeline for High-Dimensional Immune Cell Characterization.

Flow cytometry is a widely used technique for immune cell analysis, offering insights into cell composition and function. Spectral flow cytometry allows for high-dimensional analysis of immune cells, overcoming limitations of conventional flow cytometry. However, analyzing data from large antibody panels can be challenging using traditional bi-axial gating strategies. Here, we present a novel analysis pipeline designed to improve analysis of spectral flow cytometry. We employ this method to identify rare T cell populations in aging. We isolated splenocytes from young (2-3 months) and aged (18-19 months) female mice then stained these with a panel of 20 fluorescently labeled antibodies. Spectral flow cytometry was performed, followed by data processing and analysis using Python within a Jupyter Notebook environment to perform batch correction, unsupervised clustering, dimensionality reduction, and differential expression analysis. Our analysis of 3,776,804 T cells from 11 spleens revealed 34 distinct T cell clusters identified by surface marker expression. We observed significant differences between young and aged mice, with certain clusters enriched in one age group over the other. Naïve, effector memory, and central memory CD8+ and CD4+ T cell subsets exhibited age-associated changes in abundance and marker expression. Additionally, γδ T cell clusters showed differential abundance between age groups. By leveraging high-dimensional analysis methods borrowed from single-cell RNA sequencing analysis, we identified age-related differences in T cell subsets, providing insights into the immune aging process. This approach offers a robust, free, and easily implemented analysis pipeline for spectral flow cytometry data that may facilitate the discovery of novel therapeutic targets for age-related immune dysfunction.

Clonally expanded memory CD8+ T cells accumulate in atherosclerotic plaques and are pro-atherogenic in aged mice.

Aging is a strong risk factor for atherosclerosis and induces accumulation of memory CD8+ T cells in mice and humans. Biological changes that occur with aging lead to enhanced atherosclerosis, yet the role of aging on CD8+ T cells during atherogenesis is unclear. In this study, using femle mice, we found that depletion of CD8+ T cells attenuated atherogenesis in aged, but not young, animals. Furthermore, adoptive transfer of splenic CD8+ T cells from aged wild-type, but not young wild-type, donor mice significantly enhanced atherosclerosis in recipient mice lacking CD8+ T cells. We also characterized T cells in healthy and atherosclerotic young and aged mice by single-cell RNA sequencing. We found specific subsets of age-associated CD8+ T cells, including a Granzyme K+ effector memory subset, that accumulated and was clonally expanded within atherosclerotic plaques. These had transcriptomic signatures of T cell activation, migration, cytotoxicity and exhaustion. Overall, our study identified memory CD8+ T cells as therapeutic targets for atherosclerosis in aging.