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

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 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.

Extended Data Fig. 1. Aging leads to expansion of memory CD8+ T cells in circulation.

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.

Fig. 1:. CD8⁺ T cell depletion reduces atherosclerotic plaque size in Aged WT mice but not young WT mice.

(A) Schematic of murine PCSK9-AAV-induced hypercholesterolemia model of atherosclerosis and paradigm showing PCSK9 treatment protocol in young and aged female C57BL/6N WT mice. (B) CD8⁺ immunostaining in cross-section of one leaflet of the aortic sinus in young and aged C57BL/6N atherosclerotic mice with quantification of the number of CD8⁺ T cells normalized to total plaque area. (C) Schematic of murine PCSK9-AAV-induced hypercholesterolemia model of atherosclerosis and paradigm showing PCSK9 treatment protocol in C57BL/6N WT mice with anti-CD8 and anti-IgG antibody injection every 2 weeks during the WD feeding period. Representative flow cytometry plots demonstrate the reduction of CD8⁺ T cells in anti-CD8 treated mice compared to isotype control, and a line graph of serial flow cytometry data on the blood of mice demonstrates the mean reduction in CD8⁺ T cells in the anti-CD8 treatment group compared to the isotype control treatment group. Representative histology and atherosclerotic plaque size and necrotic core size quantification of the aortic sinus of young PCSK9-AAV (D), aged PCSK9-AAV (E), and Ldlr^−/− mice (F) treated with either anti-CD8 antibody treatment or anti-IgG treatment. Dashed lines indicate the atherosclerotic plaque outline and dotted lines indicate the acellular necrotic core. Scale bar = 100μm. In B, each point is a biological replicate and measurements were taken from distinct samples. In B and D-F, data are pooled from 3 independent experiments and all data are shown. In C, data were independently replicated in 3 experiments and one experiment is shown. In B and D-F, for young anti-IgG N=10, for young anti-CD8 N=11, for aged anti-IgG N=17, for aged anti-CD8 N=16, for young Ldlr^−/− anti-IgG N=7, for young Ldlr^−/− anti-CD8 N=6 over 3 independent experiments. In C, N=5 per group over 3 independent experiments (only 1 experiment shown). Data in this figure represents mean +/− SEM. 2-tailed Students T test was used for B. 2-Way ANOVA was used for C and D-F with Tukey’s post-hoc test. * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001. CM = central memory, EM = effector memory, PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease.

Fig. 2:. Aged CD8⁺ T cells contain more exhausted memory cells and are more pro-atherogenic compared to young CD8⁺ T cells.

(A) schematic of CD8⁺ T cell harvest from female young (3-mo) and female aged (18-mo) C57BL/6N WT donor spleens. Single-cell suspensions from young or aged female donors were pooled, purified for CD8⁺ T cells and then 10 million young or aged donor CD8⁺ T cells were intravenously injected into young female Cd8^−/− recipients, and a “no-cell” group received sterile PBS intravenously. Mice were allowed to recover for 1-month post-adoptive cell transfer prior to PCSK9-AAV transfection and 10-week WD feeding in all 3 groups. (B) Pie chart demonstrating the composition of enriched cell suspensions determined by flow cytometry as a percentage of all cells that were then injected into Cd8^−/− recipients. (C) Representative hematoxylin and eosin histology of the aortic sinus of Cd8^−/− mice that received either vehicle (i.e., no cells), CD8⁺ T cells from young donor spleens, or CD8⁺ T cells from aged donor spleens. Scale bars = 100μm. (D) Quantification of aortic sinus plaque size and necrotic core size of the 3 groups. 30 individual sections are analyzed from each mouse. (E) Immunohistochemical staining for anti-CD8 in tissue sections of the aortic sinus with arrowheads depicting positively stained CD8⁺ T cells with quantification to the right. Scale bars = 50μm. For B, all donor cells are pooled in each group from N=10 donors/age group over 2 independent experiments. For D and E, N=8 young Cd8^−/− + aged CD8 cells, N=13 young Cd8^−/− + young CD8 cells, and N=7 young Cd8^−/−+ no CD8 cells over 2 independent experiments. In E, each point is a biological replicate and measurements were taken from distinct samples. Data in this figure represents mean +/− SEM. For D, 2-way ANOVA with Tukey’s post-hoc test. For E, 1-way ANOVA with Tukey’s post-hoc test. CM = central memory, PD1 = programmed cell death protein 1, EM = effector memory, PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease.

Fig. 3.. Aging leads to expansion of memory CD8 cells that infiltrate aortic atherosclerotic plaque.

(A) Overview of the single cell analyses of the healthy young (2–3 month) and aged (18–19 month) female C57BL/6N WT spleens enriched for CD3⁺ T cells, and young (4–5 month) and aged (21–22 month) female C57BL/6N mPCSK9-AAV aortic plaques enriched for CD3⁺ T cells. (B) UMAP (uniform manifold approximation projection) clustering of all cells from the spleen and aorta of C57BL/6N mice showing unique cellular populations. (C) UMAP clustering of all cells split by group from the young spleens (pool of N=3 biological replicates with 18,412 total cells), aged spleens (pool of N=3 biological replicates with 11,151 total cells), young aortas (pool of N=4 biological replicates with 1,999 total cells), and aged aortas (pool of N=4 biological replicates with 5,698 total cells), color-coded by cluster identification from B. (D) Proportion of all clusters (left) and CD8⁺ T cell clusters (right) represented in each group and tissue type based on single-cell RNA seq. (E) Fold changes in cluster proportions comparing aged to young (higher fold change = increased in aged versus young) for spleen and aortic plaque. Data in this figure are from 1 independent experiment. EM = effector memory, CM = central memory, GZMK = granzyme K, LDL = low-density lipoprotein, PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease, AAV = adeno-associated virus, UMAP = uniform manifold approximation projection.

Fig. 4.. CD8 T cell populations in atherosclerotic plaques are more differentiated, exhausted, and cytotoxic with aging.

(A) UMAP scattergram of splenic and aortic CD8_Naive, CD8_CM, CD8_EM_Gzmk, CD8_EM clusters, color coded by cluster. (B) UMAP scattergram of the same clusters from A but color-coded by group. (C) UMAP scattergram of the same 4 clusters from A, but color coded by pseudotime trajectory analysis score with the cells with highest pseudotime score indicated by dashed line oval. (D) Volcano plot showing differential gene expression comparing CD8_CM and CD8_EM_Gzmk clusters with the highest pseudotime score (inside the dashed oval in C) to those with lower pseudotime scores (outside the dashed oval in C). Positive values in gene expression indicate increased expression in the cells with the highest pseudotime values. (E) UMAP scattergrams of pseudotime trajectory score split by cluster for CD8_Naive, CD8_CM, CD8_EM_Gzmk, CD8_EM with dashed oval indicating cells with the highest pseudotime trajectory values. (F) UMAP scattergrams of pseudotime trajectory score split by sample group for Young_Spleen, Aged_Spleen, Young_Aorta, Aged_Aorta with dashed oval indicating cells with the highest pseudotime trajectory values. (G) Signature score violin plot calculated for the 4 clusters from A, split by cluster and sample group, for exhaustion score (genes: Tim3, Lag3, Eomes, Pdcd1, Cd160, Tox), cytotoxicity score (genes: Ctla4, Prf1, Gzmb, Gzmk, Tigit, Ifng, Tnfrs1a), Senescence score (genes: Cdkna1, Cdkna2), and SenMayo score. For A-G, clustering of all cells split by group from the young spleens (N=3 biological replicates), aged spleens (N=3 biological replicates), young aortas (N=4 biological replicates), and aged aortas (N=4 biological replicates). For D, Venice method within BBrowser3 was used to compare groups (see Methods). Data in this figure are fromn one independent experiment. AU = arbitrary units, CM = central memory, EM = effector memory, Gzmk = granzyme K, UMAP = uniform manifold approximation projection.

Fig. 5:. Granzyme K⁺ CD8⁺ effector memory and CD8⁺ central memory cells from aged mice are clonally expanded compared to young mice.

Single-cell T cell receptor (TCR) sequencing of naive, central memory, effector memory, and granzyme K⁺ effector memory CD8⁺ T cells from disease-free 3-month old female C57BL/6N WT and 18-month old female C57BL/6N WT spleens and from atherosclerotic plaques from 3-month old C57BL/6N WT and 18-month old C57BL/6N WT mice after enriching for CD3⁺ T cells. (A) The percent of cells per unique clonotype and the number of unique clonotypes with at least 2 cells are shown. Each color represents a unique clonotype. Data are represented as a percentage of the total size of the sample. The 2 largest clonotypes, identified in within the aged plaque CD8_EM_GZMK cluster are shown in black color on the UMAP scattergram which depicts the 4 clusters of CD8⁺ T cells from all groups. (B) CDR3 sequence, V_gene, J_Gene, and clone size (number of cells) are shown along with a pie chart showing the group composition and cluster composition that makes up each of the top 5 largest clones within the CD8⁺ T cell clusters. N=3 biological replicates per group for spleen and N=4 biological replicates per group for aortic plaque. Data in this figure are from 1 independent experiment. CM = central memory, EM = effector memory, Gzmk = granzyme K, Y = young, A = aged, UMAP = uniform manifold approximation projection.

Fig. 6:. Chemokine receptors are upregulated in CD8⁺ T cell subsets in aged mice compared to young mice.

(A) Flow cytometry gating strategy for T cells from the blood of 3-mo old female C57BL/6N and 18-mo old female C57BL/6N WT mice to determine chemokine receptor expression of CCR5, CXCR3, and CXCR6. (B) Representative histograms of CCR5, CXCR6, and CXCR3 expression at baseline from 3-month old C57BL/6N WT and 18-month old C57BL/6N WT mice. Quantification of CCR5 expression on CD8⁺ CM T cells (C), CXCR6 expression on CD8⁺ CM t cells (D), and CXCR3 expression on CD8⁺ EM T cells from blood of 3-mo old C57BL/6N and 18-mo C57BL/6N female mice from baseline through 9-weeks of PCSK9-AAV treatment with western diet feeding. N=7 biological replicates per group for 3-mo old C57BL/6N and N=7 biological replicates per group for 18-mo C57BL/6N. Data in this figure are from 2 independent experiments. Data in this figure represents mean +/− SEM. For C-E, 2-way ANOVA with Šídák’s post-hoc test. AAV = adeno-associated virus, CCR5 = C-C chemokine receptor type 5, CXCR3 = C-X-C motif chemokine receptor 3, CXCR6 = C-X-C motif chemokine receptor 6, CM = central memory, EM = effector memory, MFI = median fluorescence intensity, PCSK9 = proprotein convertase subtilisin/kexin type 9 serine protease, Y = young, A = aged, WD = western diet.