The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing

Abstract

Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8⁺ T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4⁺ T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4, CCL5, etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

Keywords: Bioinformatics; Immunology.

Fig. 1. Single-cell analysis of PBMCs from patients with COVID-19.

a The cartoon outlines the study design. PBMC and BALF cells from COVID-19 patients and healthy controls were collected for scRNA-seq characterization using the 10× Genomics platform. The number of samples, analyzed cells, and samples with PBMC and BALF cells simultaneously collected from the same patient are indicated. HC, healthy controls; Mild, mild patients; Severe, severe patients. b The UMAP projection of the combined PBMC scRNA-seq dataset identifies nine major cell types. PC, plasma cells. c The specific markers for identifying each immune cell types in b are indicated. (Pct.Exp. indicates percentage of cells expressed). d Density plots show the UMAP projection of PBMCs from COVID-19 patients and controls. e The bar plot shows the proportions of each cell types in PBMCs from individual subjects. The cell numbers and ratios of monocyte/T cells are listed to the right side. f Comparisons of percentages of each cell types in PBMCs (cycling cells were re-clustered into T and PC subsets) between the two COVID-19 groups and controls (two-sided Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. H, healthy controls; M, mild patients; S, severe patients.

Fig. 2. Single-cell analysis of peripheral myeloid cell compartments in patients with COVID-19.

a UMAP plot of the five types of myeloid cells in PBMCs. b Density plots show the UMAP projection of peripheral myeloid cells from COVID-19 patients and controls. c Comparisons of percentages of each peripheral myeloid cell types between the two COVID-19 groups and controls (two-sided Student’s t-test, *P < 0.05, **P < 0.01). d Enrichment of GO biological process (BP) terms for upregulated genes (left) and downregulated genes (right) in blood CD14⁺ monocyte comparisons between mild and HC (M vs H), severe and HC (S vs H), and severe and mild groups (S vs M) (representative terms are shown, adjusted P < 0.01 as indicated by the colored bar). e The heatmaps show the selected differentially expressed genes and their associated GO terms as indicated in d (logFC > 0.41 or < –0.41, adjusted P < 0.01). Mean.Exp, average expression. f Density plots show the composite MHC II signature scores and calprotectin signature scores of peripheral CD14⁺ monocytes in 2D maps. The horizontal and vertical lines separating the four quadrants represent the median scores of all CD14⁺ monocytes. The percentages of cells in each quadrant are indicated. g Left panel shows the representative flow cytometric data of HLA-DR expression on CD14⁺ and CD14^– PBMCs. Right plot shows the summarized data from more subjects (two-sided Student’s t-test). h The Pearson correlation of “MDSC-like signature score” and plasma CRP, IL-6 levels, blood neutrophil, CD3⁺, CD4⁺, and CD8⁺ T cell counts.

Fig. 3. Abnormally activated lung monocyte-macrophages in severe COVID-19.

a The myeloid cell data from two mild and five severe COVID-19 patients who had paired PBMC and BALF samples were integrated and presented on the UMAP. b The expression of monocyte-macrophage markers FCN1, SPP1, and FABP4 were projected to UMAP from a. PM, peripheral cells of mild cases; PS, peripheral cells of severe cases; BM, BALF of mild cases; BS, BALF of severe cases. c Differentiation trajectory of the blood monocytes and BALF monocyte-macrophages from two representative COVID-19 patients, analyzed independently. d Venn diagram shows the number of upregulated and downregulated DEGs in monocyte-macrophage comparisons as indicated. logFC > 0.41 or < –0.41, adjusted P < 0.01. e Enrichment of GO biological process (BP) terms for upregulated genes (left) and downregulated genes (right) in monocyte-macrophage comparisons as indicated. Selected terms are shown, adjusted P value is indicated by the colored bar. f Heatmaps show the expression of selected interferon, cytokine, and chemokine genes in paired blood and BALF monocyte-macrophages derived from the same patient. Stars indicate that the genes are differentially expressed in BALF monocyte-macrophages between mild and severe COVID-19. Purple and green stars show that the gene expression are significantly upregulated in severe COVID-19 and mild COVID-19 groups, respectively (MAST; P < 0.01). B, BALF samples; P, PBMC samples. g The levels of selected cytokines and chemokines in paired BALF and plasma samples were measured by CBA (two-sided Wilcoxon test between BALF and PBMC of severe patients).

Fig. 4. Single-cell analysis of peripheral NK and T cell compartments in patients with COVID-19.

a UMAP plot of the 18 subsets of NK and T cells in PBMCs. b Pseudo-time differentiation trajectory of the peripheral CD4⁺ and CD8⁺ T cell subsets performed by slingshot. The bar plots in the corner shows the percentages of clonally expanded cells in each T cell subsets. c Density plots show the UMAP projection of peripheral NK and T cells from COVID-19 patients and controls. d Comparisons of percentages of each peripheral NK and T cell types between the two COVID-19 groups and controls (two-sided Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001). e Clonally expanded T cells from COVID-19 patients and controls are projected into UMAP from a. f Clonal expansion indexes of T cell subsets from COVID-19 patients and controls are separately displayed. g T cell state transition status among any two clusters is inferred by their shared TCR clonotypes. Each T cell cluster is represented by a unique color. The numbers above the bar indicate the percentages of cells sharing TCRs in those two clusters.

Fig. 5. Tracking T cells across peripheral blood and BALFs in patients with COVID-19.

a The T cell data from two mild and five severe COVID-19 patients who had paired PBMC and BALF samples were integrated and presented on the UMAP. b The percentages of each T cell subset in paired BALF (B) and PBMC (P) of the same patient are compared. c Heatmaps display the selected DEGs in NK, CD4-Tm, and CD8-Tm cells in BALF (B) or PBMC (P) from the mild (M) or severe (S) COVID-19 patients. Cytokine-related genes are red marked (logFC > 0.41, adjusted P < 0.01). d The migration index in each T cell subset across paired PBMC and BALF from seven patients are shown (STARTRAC-migr indices). e TCR clonotypes were classified into five different types as indicated by different color bars (singleton indicates non-expanded TCR clonotype, multiplet indicates expanded TCR clonotype, dual-clone indicates those clonotype shared in paired PBMC and BALF samples). The bar plots show the percentages of different types of TCR clonotypes in different T cell subsets from paired PBMC and BALF samples. f The circus plot shows the degree of TCR clonotype sharing across different T cell subsets in PBMC and BALF from the mild and severe COVID-19 groups. g Heatmap shows the selected DEGs in each T cell clones derived from the top 13 TCR clonotypes shared across PBMC vs BALF compartments (logFC > 0.41, adjusted P < 0.01). h The V, J genes of the TCR α and β chains of the top 13 dual clonotypes are listed, and the amino acid sequences of their CDR3 are shown.