To generate a comprehensive immune cell atlas reflecting cellular and systemic adaptations resulting from age and/or COVID-19 infection, we integrated scRNA-seq, CyTOF, scATAC-seq and scTCR/BCR-seq of single-cell PBMC suspensions collected from 3 separate cohorts (Fig. 1A, 1B, and Table S1A–G). In cohort-1, comprising young healthy adults (YA) (20–45 years old) and aged healthy adults (AA) (≥60 years old), we combined CyTOF (n = 10) and scATAC-seq (n = 10) with scRNA-seq (n = 16) and scTCR/BCR-seq (n = 16); in cohort-2, comprising young healthy (YH) individuals (30–45 years old), aged healthy (AH) individuals (≥60 years old), young COVID-19 onset patients (YCO) (30–50 years old) and aged COVID-19 onset patients (ACO) (≥70 years old), we performed CyTOF analysis (n = 8); and in cohort-3, comprising YH individuals, AH individuals, young recovered COVID-19 patients (YCR) (30–50 years old) and aged recovered COVID-19 patients (ACR) (≥70 years old), we performed scRNA-seq (n = 22) (Fig. 1B). By combining scRNA-seq, CyTOF, scATAC-seq and scTCR/BCR-seq analysis, we created a comparative framework detailing the impact of aging on cell type distribution and immune cell functions at the transcriptional, proteomic, and chromatin accessibility levels in cohort-1. In cohort-2, we measured single-cell protein expression using a 26-marker CyTOF panel to discover early cellular changes in incipient COVID-19 patients and how those changes were affected by age. Finally, in cohort-3, we compared cellular differences between young and aged recovered COVID-19 patients by scRNA-seq analysis (Fig. 1B).
Figure 1 Schematic illustration of the collection and data processing of PBMC from young and aged group. (A) Flowchart overview of PBMC collection in young and aged adults followed by scRNA-seq, mass cytometry, scATAC-seq and scTCR/BCR-seq experiments. (B) Schematic illustration of experimental cohorts; cohort-1: young and aged adults, cohort-2: young and aged healthy individuals, young and aged adults with COVID-19 onset, cohort-3: young and aged healthy individuals, young and aged adults recovered from COVID-19, matched with analysis as indicated: single-cell proteomic data from CyTOF studies, gene expression data from scRNA-seq studies, chromosomal accessibility data from scATAC-seq, and TCR and BCR repertoire data from scTCR/BCR-seq. (C) t-SNE projections of PBMCs derived from scRNA-seq data in cohort-1. (D) Heatmaps showing scaled expression of discriminative gene sets for each cell type and cell subset. Color scheme is based on z-score distribution from −3 (purple) to 3 (yellow)