Dysregulated metal ion homeostasis underscores non-canonical function of CD8+ T cell during COVID-19

Abstract

Introduction: Several efforts have been made to describe the complexity of T cell heterogeneity during the COVID-19 disease; however, there remain gaps in our understanding in terms of the granularity within.

Methods: For this attempt, we performed a single-cell transcriptomic analysis of 33 individuals (4 healthy, 16 COVID-19 positive patients, and 13 COVID-19 recovered individuals).

Results: We found CD8⁺ T cell-biased lymphopenia in COVID-19 patients compared to healthy and recovered individuals. We also found an optimal Th1/Th2 ratio, indicating an effective immune response during COVID-19. Expansion of activated CD4⁺ T and NK T was detected in the COVID-19-positive individuals. Surprisingly, we found cellular and metal ion homeostasis pathways enriched in the COVID-19-positive individuals compared to the healthy and recovered in the CD8⁺ T cell populations (CD8⁺ TCM and CD8⁺ TEM) as well as activated CD4⁺ T cells.

Discussion: In summary, the COVID-19-positive individuals exhibit a dynamic T cell mediated response. This response may have a possible association with the dysregulation of non-canonical pathways, including housekeeping functions in addition to the conventional antiviral immune response mediated by the T cell subpopulation. These findings considerably extend our insights into the heterogeneity of T cell response during and post-SARS-CoV-2 infection.

Keywords: COVID-19; T cell heterogeneity; metal ion; non-canonical; single cell RNA-seq.

Figure 1

Study outline, workflow of experiment and hypothesis testing. (A) Study outline and experimental workflow, comprising sample collection and grouping, PBMC isolation, single-cell sequencing, data analysis, and interpretation. (B) Hypothesis toward investigating the functional role of T cell heterogeneity in SARS-CoV-2 infected individuals.

Figure 2

UMAP visualization of peripheral blood immune cells and distribution of T cell subsets in healthy, positive, and recovered individuals. (A) Graphical representation of the panel outline. (B) UMAPs of 124,476 single cells with 17 cell types annotated from single-cell sequencing of PBMCs from the individuals. (C–E) UMAPs of eight T cell subsets in healthy, COVID-19 positive and recovered individuals, respectively. T cell subsets are colored with their respective colors, while the rest of the cell types are gray in color. (F) Radial plot with T cell abundance compared to other cells in healthy, positive, and recovered individuals. (G) A circular bar chart showing the relative abundance of T cell subsets across healthy, positive, and recovered; significance calculated by the Fisher exact test (p > 0.05: ns, p < 0.05 > 0.01: *, p < 0.01 > 0.001: **, p < 0.001 > 0.0001: ***, p < 0.0001: ****).

Figure 3

Differential gene expression of T cells. (A) Graphical representation of the panel outline. (B–D) Volcano plot depicting all the differentially expressed genes in clustered pairwise comparison (Healthy vs. Positive. Positive vs. Recovered, and Healthy vs. Recovered) across the T cell population. The green dots describe genes with log2 fold change more than ±1.5; the red dots represent significant genes based on both q-value and log2 fold change. (E) Pearson correlation plot showing the significant differentially expressed genes across the pairwise comparison groups. The boxes denote genes that are differentially expressed and have a positive correlation score that are grouped together. Asterisk marked clusters are found to be significant in the Pearson correlation analysis (p-value ≤ 0.05).

Figure 4

Cluster and T cell specific pathways in COVID-19-patients. (A) Dot Plot depicting upregulated DEGs and cluster specific enriched GO pathways in COVID-19 positive compared to healthy individuals. (B) Dot Plot depicting upregulated DEGs and cluster specific enriched GO pathways in the COVID-19 positive patients when compared with recovered individuals. (C) Sankey plots indicating cluster 14 derived significant pathways enriched in CD8⁺ TCM, CD8⁺ TEM, and activated CD4⁺ T cell types. (D) Sankey plots indicating clusters 14, 5, and 15 derived significant pathways enriched in CD8⁺ TEM, activated CD4⁺ T, Th1, CD8⁺ TCM, NK T, and Th2 cell types. Pathways are highlighted according to their broad categories; immune related are in red, homeostasis are in green, stress response are in purple; and metabolic response are in brown.

Figure 5

Summary highlighting the Single cell RNA-seq based T cell heterogeneous response in COVID-19 positive individuals.