Sex Differences in Coronary Artery Disease and Diabetes Revealed by scRNA-Seq and CITE-Seq of Human CD4+ T Cells

Abstract

Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD−. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.

CITE-seq analysis reveals human cytomegalovirus and diabetes-associated adaptive NK cell alterations in cardiovascular disease

“…To study the relationship between CAD and NK cells in humans, we analyzed PBMC CITE-seq data from a cohort of 61 patients diagnosed with a low or high CAD severity score (percent stenosis of each artery segment; CAD low : 0-6, or CAD high : x > 30, and as was previously defined: GSE190570) 22 . Thirty-one patients were diagnosed with T2DM (type 2 diabetes).…”

“…Next, the tubes containing the PBMCs were centrifuged at 400 × g for 5 minutes. The cells were then resuspended in a combination of 51 AbSeq antibodies, with each antibody added at a volume of 2 μL and 20 μL of BD’s Stain Buffer solution 22 . This resuspension process was per-formed on ice for 30-60 minutes according to the manufacturer’s recommendations.…”

“…The resulting FASTA and FASTQ files were uploaded to the Seven Bridges Genomics pipeline (https://www.sevenbridges.com/apps-and-pipelines/, accessed on 9 November 2020), where data filter-ing was applied to generate matrices and CSV files. This analysis yielded draft transcriptomes and surface phenotypes of 213,515 cells involving 496 genes and 51 antibodies 22 . After removing cell doublets based on sample tags and undetermined cells, 175,628 cells remained.…”

Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease

“…Coronary angiography allows us to apply a well-validated quantitative atherosclerosis severity score, known as the Gensini score, that has proven to be highly effective in predicting the risk of future cardiovascular events. Subjects within our study were stratified based on Gensini scores as either having low CAD (Gensini scores ≤ 6) or high CAD (Gensini scores > 32) [ 12 , 13 ]. We included both males and females in our study, and all statistical analyses were adjusted for statin use, smoking, and diabetes as covariates.…”

“…Recent studies of ours and others, using high dimensional immunoprofiling methods, have identified multiple immune cells present in the mouse atherosclerotic aorta, including previously unknown immune cell subclusters [ 10 , 11 ]. In human clinical atherosclerosis, we and others have reported changes in human peripheral blood T and B cells [ 12 , 13 ] and plaque-localized immune cells and macrophages [ 14 , 15 , 16 ]. We have reported detailed phenotypic heterogeneity in human peripheral blood monocytes from healthy individuals [ 17 ].…”