Abstract
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 female 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.
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Data availability
Raw and processed mouse single-cell RNA sequencing and TCR sequencing data are deposited in the Gene Expression Omnibus (GSE210719). Genome builds used can be found at https://support.10xgenomics.com/single-cell-gene-expression/software/release-notes/build. Human atherosclerotic plaque data were re-analyzed from publicly available data archived on Zenodo: 3361716. The source data figures in this manuscript are published along with the manuscript. Raw data are available from the corresponding author upon reasonable request.
Code availability
No original code was written for the experiments in this study. The packages for the code used are detailed in the Methods subsection titled ‘scRNA-seq’.
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Acknowledgements
This study was supported by National Institutes of Health (NIH) awards AG068309 (D.J.T.), HL155169, AG028082, AI138347 (D.R.G.), HL158003 (J.C.) and AHA898210 (J.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We acknowledge support from the Bioinformatics Core of the University of Michigan Medical School’s Biomedical Research Core Facilities, especially D. King. We acknowledge W. Rosebury-Smith in the Unit for Laboratory Animal Management, In-Vivo Animal Core, for expertise and assistance with histology. Research reported in this publication was supported by the National Cancer Institute under award P30CA046592 by use of the following Cancer Center Shared Resource: Single Cell and Spatial Analysis Shared Resource.
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D.J.T. and D.R.G. conceived the project and performed experiments. D.J.T. and D.R.G. secured funding. D.J.T. and D.R.G. analyzed the data. D.J.T. and D.R.G. wrote the manuscript. All authors edited the manuscript.
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Nature Aging thanks Klaus Ley, Tin Kyaw and Holger Winkels for their contributions to the peer review of this work.
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Extended data
Extended Data Fig. 1 Aging leads to expansion of memory CD8+ T cells in circulation.
(a) Schematic of experimental procedure to isolate blood and perform flow cytometry at 3 timepoints during atherogenesis in young wild-type, aged wild-type, and young Ldlr-/- mice. (b) Blood-cell flow cytometry gating strategy to identify CD8+ T cells. Live CD45+ lymphocytes were gated on CD3e+ cells, CD8+ cells, and subdivided into naive (CD62L+CD44−), central memory (CD62L+CD44+), and effector memory (CD44+CD62L−) then PD1+ and either granzyme K+ or Tox+ cells. (c-l) Flow cytometry quantification of T cell populations as a frequency of CD3+ T cells for young C57BL/6, aged C57BL/6, and young Ldlr-/-, mice at baseline, midway through the western diet feeding period (5-weeks), and just prior to sacrifice (10-weeks). N = 6 biological replicates per group over 1 independent experiment. Measurements were taken from distinct samples. Data in this figure represents mean ± SEM. 2-Way ANOVA with Tukey’s post-hoc test. PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease, AAV = adeno-associated virus, CM = central memory, EM = effector memory.
Extended Data Fig. 2 Aging leads to expansion of memory CD8+ T cells in circulation.
Representative flow cytometry plots of T cells for young WT, aged WT, and young Ldlr-/- mice after PCSK9-AAV and 10-weeks of western diet feeding. Representative flow cytometry plots demonstrating increased frequency of central memory CD8+ T cells in aged C57BL/6 WT mice and young Ldlr-/- mice compared to young C57BL/6 WT mice, increased frequency of effector memory CD8+ T cells in aged C57BL/6 WT mice versus young C57BL/6 WT and Ldlr-/- mice, and greater frequency of naive CD8+ T cells in young C57BL/6 WT mice and young Ldlr-/- mice compared with aged C57BL/6 WT mice. Aged WT mice also demonstrate greater frequency of PD1+ EM CD8+ T cells and granzyme K+ PD1+ EM CD8+ T cells compared to young WT and Ldlr-/- mice. PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease, AAV = adeno-associated virus, CM = central memory, EM = effector memory.
Extended Data Fig. 3 Similar fasting total cholesterol level in young and aged C57BL/6 WT mice treated with anti-IgG or anti-CD8 treatment over 10-weeks of WD feeding.
Fasting total cholesterol was quantified from plasma via colorimetric assay. 2-way ANOVA with Tukey’s post-hoc test. N = 11 young WT anti-CD8a mice, N = 10 young WT anti-IgG mice, N = 16 aged WT anti-CD8 mice, N = 17 aged WT anti-IgG mice, N = 6 young Ldlr-/- anti-IgG mice, and N = 7 young Ldlr-/- anti-CD8 mice over 3 biological replicates. Data in this figure represents mean ± SEM. Measurements were taken from distinct samples. Ldlr = low-density lipoprotein receptor. WD = western diet, WT = wild-type.
Extended Data Fig. 4 CD8+ T cell depletion reduces brachiocephalic artery atherosclerotic plaque size in Aged WT mice but not young WT mice.
Murine PCSK9-AAV-induced hypercholesterolemia model of atherosclerosis with anti-CD8 or anti-IgG antibody injection every 2 weeks during the WD feeding period. Representative histology and atherosclerotic plaque size quantification of the brachiocephalic artery of young PCSK9-AAV (a), aged PCSK9-AAV (b), and Ldlr-/- mice (c) treated with either anti-CD8 antibody treatment or anti-IgG treatment. Scale bar = 100 µm. In A-C, data are pooled from 3 independent experiments and all data are shown. N = 10 for young anti-IgG, N = 11 for young anti-CD8, N = 17 for aged anti-IgG, N = 16 for aged anti-CD8, N = 7 for young Ldlr-/- anti-IgG, and N = 6 for young Ldlr-/- anti-CD8 over 3 independent experiments. Measurements were taken from distinct samples. Data in this figure represents mean ± SEM. 2-Way ANOVA with Tukey’s post-hoc test. BCA = brachiocephalic artery, CM = central memory, EM = effector memory, PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease.
Extended Data Fig. 5 Anti-CD8 treatment significantly reduces the number of CD8+ T cells in atherosclerotic aortas.
Aortas from aged mice transfected with PCSK9-AAV and subjected to 10-weeks of western diet feeding and either treated with anti-CD8 or anti-IgG isotype control antibody during western diet feeding were digested and analyzed by flow cytometry. The flow cytometry gating strategy is shown demonstrating CD4+ and CD8+ T cells in anti-CD8 and anti-IgG treated atherosclerotic mice. Quantification demonstrates a significant reduction of CD8+ T cells in anti-CD8 treated mice compared to isotype control. N = 5 anti-IgG treated mice and N = 5 anti-CD8 treated mice over 1 independent experiment. Data in this figure represents mean ± SEM. Each point is a biological replicate and measurements were taken from distinct samples. Two-tailed Mann-Whitney U test.
Extended Data Fig. 6 CD8+ T cell enrichment from young and aged C57BL/6 WT donor spleens.
(a) Flow cytometry gating strategy for young and aged WT mice before and after CD8+ T cell enrichment prior to CD8+ T cell adoptive transfer into Cd8-/- mice showing gating and frequency of previous gate. (b) Representative flow cytometry plots demonstrating CD4+ and CD8+ T cells, memory and naive CD8+ T cells, PD1+ effector memory CD8+ T cells, and PD1+ central memory CD8+ T cells showing gating and frequency of parent gate.
Extended Data Fig. 7 Similar fasting total cholesterol level in young Cd8-/- mice adoptively transferred with 10 million CD8+ T cells or vehicle from either young C57BL/6 WT or aged C57BL/6 WT mice over 10-weeks of WD feeding.
All mice were adoptively transferred and allowed to rest for 4-weeks before PCSK9-AAV injection and 10-week WD feeding. Fasting total cholesterol was quantified from plasma via colorimetric assay. 2-way ANOVA with Tukey’s post-hoc test. N = 13 young Cd8-/- mice + young CD8+ T cells, N = 8 young Cd8-/- mice + aged CD8+ T cells, and N = 7 young Cd8-/- mice + no CD8+ T cells over 2 independent experiments. Data in this figure represents mean ± SEM. Measurements were taken from distinct samples. WD = western diet, WT = wild-type.
Extended Data Fig. 8 Human atherosclerotic plaque CD8 T cells express GZMK and associate with symptomatic atherosclerotic disease.
(a) UMAP plot of T cells from human atherosclerotic plaques from public study deposited to Zenodo: 3361716. (b) GZMK expression overlayed on UMAP plot of T cells in human atherosclerotic plaques with dashed line indicating cells with the highest GZMK expression. (c) top 15 differentially expressed genes in the CD8+ T cell cluster. (d) Violin plot of GZMK expression on 3 T cell clusters, stratified by symptomatic group. For D, two-tailed Venice non-parametric benchmarking method within BBrowser3 was used to compare groups (see Methods) and data represents mean with lines above and below representing 1st and 3rd quartiles. Raw data for this figure is from a publicly available dataset archived on Zenodo: 3361716. UMAP = uniform manifold approximation projection.
Extended Data Fig. 9 Atherosclerotic plaque T cells are more clonally expanded than splenic T cells and aging enhances clonal expansion.
(a) Abundance of unique clonotypes. (b) Clonotype diversity estimation by Chao1 index. (c) Length of CDR3 sequences by group. (d) Size of clonotypes by group. (e) Clonotype tracking of the top 5 clonotypes from each sample across all other samples. (f) Overlap of CDR3 sequences by sample in circos plot. Top 100 expanded CDR3 sequences, colored by group, of 15 amino acids (g), 10 amino acids (h), and 5 amino acids (i). Composition of amino acid sequences of different lengths including 15 amino acids (j), 10 amino acids (k), and 5 amino acids (l). Depiction of which samples had the greatest clonal expansion shared across the number of cells (x-axis) and number of samples (y-axis) and stratified by group (m), age (n), and tissue-type (o). Data includes young spleens (N = 3 biological replicates and 18,412 total cells), aged spleen (N = 3 biological replicates and 11,151 total cells), young aorta (N = 4 biological replicates and 1,999 total cells), and aged aorta (N = 4 biological replicates and 5,698 total cells) over 1 independent experiment.
Extended Data Fig. 10 Aged atherosclerotic mice have more CD3+, CD8+, and F4/80+ cells in the aorta compared to young mice.
Aortas from 3-mo old and 18-mo old mice transfected with PCSK9-AAV and subjected to 10-weeks of western diet feeding were digested and analyzed by flow cytometry. (a) The flow cytometry gating strategy is shown CD3+, CD8+, and CD11b+F4/80+ cells from the aortas of atherosclerotic mice. Quantification demonstrates a significant increase of CD3+ and CD8+ T cells as well as CD11b+F4/80+ macrophages in aged atherosclerotic mice compared with young atherosclerotic mice normalized to total aorta weight (b-d) or by mg of aorta weight (e-g). In B-G, each point is a biological replicate and measurements were taken from distinct samples. N = 6 aged and N = 7 young mice at each timepoint for a total of N = 18 aged mice and N = 21 young mice over 1 independent experiment. Data in this figure represents mean ± SEM. 2-Way ANOVA with Šídák’s post-hoc test. PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease, AAV = adeno-associated virus, CM = central memory, EM = effector memory, WD = western diet.
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Tyrrell, D.J., Wragg, K.M., Chen, J. et al. Clonally expanded memory CD8+ T cells accumulate in atherosclerotic plaques and are pro-atherogenic in aged mice. Nat Aging 3, 1576–1590 (2023). https://doi.org/10.1038/s43587-023-00515-w
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DOI: https://doi.org/10.1038/s43587-023-00515-w
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