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

Flow cytometry is used for immune cell analysis for 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 Ab panels is challenging using traditional biaxial gating strategies. We present, to our knowledge, a novel analysis pipeline 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 mo old) and aged (18-19 mo old) female C57BL/6N mice and then stained these with a panel of 20 fluorescently labeled Abs. We performed spectral flow cytometry and then data processing and analysis using Python within a Jupyter Notebook environment to perform dimensionality reduction, batch correction, unsupervised clustering, and differential expression analysis. Our analysis of 3,776,804 T cells from 11 spleens revealed 35 distinct T cell clusters identified by surface marker expression. We observed significant differences between young and aged mice, with clusters enriched in one age group over the other. Naive, effector memory, and central memory CD8+ and CD4+ T cell subsets exhibited age-associated changes in abundance and marker expression. We also demonstrate the utility of our pipeline in a human PBMC dataset that used a 50-fluorescent color panel. By leveraging high-dimensional analysis methods, we provide insights into the immune aging process. This approach offers a robust and easily implemented analysis pipeline for spectral flow cytometry data that may facilitate the discovery of novel therapeutic targets for age-related immune dysfunction.

Cardiovascular aging: spotlight on mitochondria.

Mitochondria are cellular organelles critical for ATP production and are particularly relevant to cardiovascular diseases including heart failure, atherosclerosis, ischemia-reperfusion injury, and cardiomyopathies. With advancing age, even in the absence of clinical disease, mitochondrial homeostasis becomes disrupted (e.g., redox balance, mitochondrial DNA damage, oxidative metabolism, and mitochondrial quality control). Mitochondrial dysregulation leads to the accumulation of damaged and dysfunctional mitochondria, producing excessive reactive oxygen species and perpetuating mitochondrial dysfunction. In addition, mitochondrial DNA, cardiolipin, and N-formyl peptides are potent activators of cell-intrinsic and -extrinsic inflammatory pathways. These age-related mitochondrial changes contribute to the development of cardiovascular diseases. This review covers the impact of aging on mitochondria and links these mechanisms to therapeutic implications for age-associated cardiovascular diseases.

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.

Tissue resident memory CD8+ T cells are present but not critical for demyelination and neurodegeneration in a mouse model of multiple system atrophy.

Multiple system atrophy (MSA) is rare, fast progressing, and fatal synucleinopathy with alpha-synuclein (α-syn) inclusions located within oligodendroglia called glial cytoplasmic inclusions (GCI). Along with GCI pathology there is severe demyelination, neurodegeneration, and neuroinflammation. In post-mortem tissue, there is significant infiltration of CD8+ T cells into the brain parenchyma, however their role in disease progression is unknown. To determine the role of CD8+ T cells, a modified AAV, Olig001-SYN, was used to selectively overexpress α-syn in oligodendrocytes modeling MSA in mice. Four weeks post transduction, we observed significant CD8+ T cell infiltration into the striatum of Olig001-SYN transduced mice recapitulating the CD8+ T cell infiltration observed in post-mortem tissue. To understand the role of CD8+ T cells, a CD8 knockout mice were transduced with Olig001-SYN. Six months post transduction into a mouse lacking CD8+ T cells, demyelination and neurodegeneration were unchanged. Four weeks post transduction, neuroinflammation and demyelination were enhanced in CD8 knockout mice compared to wild type controls. Applying unbiased spectral flow cytometry, CD103+, CD69+, CD44+, CXCR6+, CD8+ T cells were identified when α-syn was present in oligodendrocytes, suggesting the presence of tissue resident memory CD8+ T (Trm) cells during MSA disease progression. This study indicates that CD8+ T cells are not critical in driving MSA pathology but are needed to modulate the neuroinflammation and demyelination response.

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.

Kernel Water Relations and Kernel Filling Traits in Maize (Zea mays L.) Are Influenced by Water-Deficit Condition in a Tropical Environment.

Water deficit is a major limiting condition for adaptation of maize in tropical environments. The aims of the current observations were to evaluate the kernel water relations for determining kernel developmental progress, rate, and duration of kernel filling, stem reserve mobilization in maize. In addition, canopy temperature, cell membrane stability, and anatomical adaptation under prolonged periods of pre- and post-anthesis water deficit in different hybrids was quantified to support observations related to kernel filling dynamics. In this context, two field experiments in two consecutive years were conducted with five levels of water regimes: control (D1), and four water deficit treatments [V10 to V13 (D2); V13 to V17 (D3); V17 to blister stage (D4); blisters to physiological maturity (D5)], on three maize hybrids (Pioneer 30B80, NK 40, and Suwan 4452) in Expt. 1. Expt. 2 had four water regimes: control (D1), three water deficit treatments [V10 to anthesis (D2); anthesis to milk stage (D3); milk to physiological maturity (D4)], and two maize hybrids (NK 40 and Suwan 4452). Water deficit imposed at different stages significantly reduced maximum kernel water content (MKWC), kernel filling duration (KFD), final kernel weight (FKW), and kernel weight ear-1 while it increased kernel water loss rate (KWLR), kernel filling rate (KFR), and stem weight depletion (SWD) across maize hybrids in both experiments. The lowest MKWC under water deficit was at D3 in both experiments, indicating that lower KFR results in lowest FKW in maize. Findings indicate that the MKWC (R 2 = 0.85 and 0.41) and KFR (R 2 = 0.62 and 0.37) were positively related to FKW in Expt. 1 and 2, respectively. The KFD was reduced by 5, 7, 7, and 11 days under water deficit at D3, D4 in Expt. 2 and D4, D5 in Expt. 1 as compared to control, respectively. Water deficit at D5 in Expt. 1 and D4 in Expt. 2 increased KWLR, KFR, and SWD. In Expt. 2, lower canopy temperature and electrical conductivity indicated cell membrane stability across water regimes in NK 40. Hybrid NK 40 under water deficit had significantly higher cellular adaptation by increasing the number of xylem vessel while reducing vessel diameter in leaf mid-rib and attached leaf blade. These physiological adjustments improved efficient transport of water from root to the shoot, which in addition to higher kernel water content, MKWC, KFD, KFR, and stem reserve mobilization capacity, rendered NK 40 to be better adapted to water-deficit conditions under tropical environments.