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Immune cell profiling of COVID‑19 patients in the recovery stage by single-cell sequencing

On June 8th 2020, the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) or COVID‑19 global pandemic had reached 6.9 million cases of infection and over 400,000 deaths according to the WHO website. Studies on other coronavirus infections such as severe acute respiratory syndrome coronavirus (SARS‑CoV) and Middle East respiratory syndrome coronavirus (MERSCoV) have identified immune system dysregulation, such as lymphopenia and inflammatory cytokine storm, that may be associated with the severity of infection. Recent studies on SARS‑CoV‑2 found decreased lymphocytes and increased inflammatory cytokine levels in peripheral blood serum. However, the mechanisms and types of immune cell activation during COVID‑19 infection have yet to be determined.

In this study, the authors used single cell sequencing to determine changes in the transcriptome of immune cells from PBMCs of patients infected with COVID‑19 aiming to identify key changes in the activation of immune cell subtypes and identify immune clearance mechanisms that can be targeted for therapeutic treatments.

Ten COVID‑19 patients in early (ESR) or late stage recovery (LSR) were compared to five healthy volunteers using scRNA‑seq and paired scTCR and scBCR‑seq. Single cell suspensions were barcoded using the 10X Genomics platform and analysed using Cell Ranger software.

Immune cells were clustered by t-distributed scholastic neighbour embedding (t‑SNE) using the canonical markers CD14, CD1C, and FCGR3A for myeloid cells; CD3E, CD4, CD8A, and NCAM1 for NK and T cells and CD19 for B cells. Twenty clusters of immune cells were identified.

All major immune cell lineages were identified in COVID‑19 infected patients. Both ERS and LRS COVID‑19 patients had a higher proportion of myeloid cells compared to the HCs, but a lower proportion of NK and T cells. LRS had more B, T and NK cells and fewer myeloid cells than ERS indicating decreased lymphocyte and increased myeloid counts in COVID‑19 patients.

All immune cell subtypes had high levels of expression of inflammatory cytokines and chemokines compared to corresponding subtypes from healthy controls.

Monocytes were further clustered into six subsets based on expression of different marker genes. The ratio of classical monocytes (CD14++) was higher in ERS patients compared to healthy volunteers whereas in LRS ratios were similar to normal. Higher numbers of CD14++/IL1B and IFN-activated monocytes were detected in COVID‑19 patients compared to controls.

Inflammation-associated genes such as IL1β, JUN, FOS, JUNB, and KLF6, chemokines, CCL4, CXCR4 and interferon-stimulated genes, IFRD1, IRF1 and IFI6 were expressed in these monocytes whereas anti-inflammatory genes were downregulated in COVID‑19 patients compared to controls suggesting a role for these monocytes in increased inflammatory response to COVID‑19 infection.

NK and T-cell clusters were also subdivided based on expression of activated marker genes. Numbers of CD8+ T cells and in particular effector memory CD8+ T cells and NK cells were decreased in COVID‑19 patients compared to healthy volunteers.

Similar to the monocytes, CD4+ T cells expressed high levels of pro-inflammatory genes such as FOS, JUN, KLF6, and S100A8, whereas anti-inflammatory genes were downregulated.

Using TCR-seq the authors demonstrated a decrease in T-cell expansion in the ERS group compared to controls although effector memory T cells, terminal effector CD8+ T cells (CTLs) and proliferating T cells showed higher expansion levels. CD8+ CTLs demonstrated increased activation of inflammation and antiviral activity compared to those from healthy volunteers.

Four B-cell clusters were identified using BCR-seq, naïve B cells (CD19, CD20, IGHD, IGHM, IL4R, and TCL1A), memory B (CD27, CD38, and IGHG), immature B (CD19 and CD20) and plasma cells (XBP1 and MZB1).

The percentage of plasma cells were significantly increased in COVID‑19 patients and naïve B cells decreased compared to healthy volunteers. Activated B-Cell genes such as S100A8, IGLL5, SSR3, IGHA1, XBP1, and MZB1 were expressed mainly in the Memory B and plasma cells of the ERS patients

The IgA isotype was found to be over-represented in COVID‑19 patients compared to healthy controls and this correlated with serum IgA levels. (IgA+IgG+IgE) to (IgD+IgM) ratios were significantly increased in the ERS patients and decreased as patients recovered.

COVID‑19 patients had significantly expanded B-cell clones compared to healthy volunteers consistent with unique clonal VDJ rearrangements due to COVID‑19 infection. Higher clonality was detected in ERS compared to LRS.

CD27+CD38+ memory B cells showed the highest clonal expansion in the B-cell subtypes.

Differential expression of the cloned cells demonstrated increased B-cell gene expression (CD27, SSR4, IGHG1, MZB1, and XBP1) and expression of these genes decreased as patients recovered.

Usage of VDJ genes demonstrated over-representation of the IGHV3 family, especially the IGHV3‑7, IGHV3‑15, IGHV3‑21, IGHV3‑23, and IGHV3‑30 in COVID‑19 patients with ESR patients having a top pairing of IGHV3‑23‑IGHJ4 and IGHV3‑7‑IGHJ6.

In this study, one of the first describing potential mechanisms for the more extreme symptoms observed in COVID‑19 patients, the authors applied single cell sequencing to characterise the immune cell repertoire and gene expression changes in recovering patients. The results demonstrated a sustained inflammatory response in patients even in late stage recovery especially in monocytes where IL1B+ were the major subsets. Importantly, B and T-cell clones were highly expanded in COVID‑19 patients compared to controls and the authors identified biased usage of IGV genes, IGHV3‑23 and IGHV3‑7, which could provide a framework for vaccine design.

Reference

Wen et al, 2020. Immune cell profiling of COVID‑19 patients in the recovery stage by single-cell sequencing Cell Discovery 6 Article number: 31

https://doi.org/10.1038/s41421-020-0168-9

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