Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Landscapes of SARS-CoV-2-reactive CD8+ T cells: heterogeneity of host immune responses against SARS-CoV-2

In a recent study published in Science Immunology,1 Kusnadi et al. performed single-cell RNA sequencing (scRNA-seq) of SARS-CoV-2-reactive CD8+ T cells and reported heterogeneity.

SARS-CoV-2 infection causes COVID-19, which is an ongoing pandemic disease threatening public health. The virology of SARS-CoV-2 and immune responses against the virus have been urgently investigated to develop effective measures against COVID-19. During viral infection, CD8+ T cells contribute to elimination of the virus by exerting cytotoxicity against virus-infected cells and producing effector cytokines, whereas neutralizing antibodies interfere with viral entry of host cells.

After the emergence of COVID-19, early studies examined the phenotypes and functions of various subtypes of immune cells from infected patients using high-dimensional techniques, including scRNA-seq and multi-parameter cytometry.2,3 These studies also revealed the profiles of CD8+ and CD4+ T cells in patients with COVID-19. However, the data did not include information regarding virus-specificity of T cells because these studies analyzed total CD8+ or CD4+ T cells, not SARS-CoV-2-reactive CD8+ or CD4+ T cells.

Other studies have detected and characterized SARS-CoV-2-reactive CD8+ and CD4+ T cells using ex vivo antigen stimulation-based assays, including interferon (IFN)-γ ELISpot assays, intracellular cytokine staining (ICS), and activation-induced marker (AIM) assays.4 Intriguingly, SARS-CoV-2-reactive CD8+ and CD4+ T cells have been detected not only in COVID-19 patients and convalescents, but also unexposed individuals. MHC class I (MHC-I) multimers were also used to directly detect SARS-CoV-2-specific CD8+ T cells without ex vivo stimulation, and their phenotypes were examined among COVID-19 patients and convalescents.5 Although these studies examined the phenotypes and functions of SARS-CoV-2-reactive T cells, high-dimensional techniques, such as scRNA-seq, could not be combined; thus, the deep profiles of SARS-CoV-2-reactive T cells have not been elucidated.

In a recent study, Kusnadi et al. examined landscapes of the SARS-CoV-2-reactive CD8+ T-cell population in a comparison with influenza A virus (IAV)-reactive and respiratory syncytial virus (RSV)-reactive CD8+ T-cell populations by scRNA-seq analysis.1 First, they isolated each virus-reactive CD8+ T-cell population from the peripheral blood mononuclear cells (PBMCs) of patients with COVID-19, or healthy donors via modified antigen-reactive T-cell enrichment (ARTE) (Fig. 1). In modified ARTE, PBMCs were stimulated ex vivo for 24 h with overlapping peptide pools for each viral protein, and responding CD8+ T cells were isolated based on the expression of activation markers CD137 and CD69. Next, they performed scRNA-seq analysis of each viral protein-reactive CD8+ T-cell population.

Fig. 1: Single-cell RNA sequencing of virus-specific CD8+ T cells.

a Total CD8+ T cells include CD8+ T cells with various antigen specificity. b, c Virus-specific CD8+ T cells are fluorescently detected by activation-induced markers, such as CD69 and CD137, following ex vivo stimulation with viral antigens (b) or MHC-I multimer staining (c). d Virus-specific CD8+ T cells are enriched by sorting fluorescently stained cells. The procedure enriching activation-induced marker+ cells is called antigen-reactive T-cell enrichment (ARTE). e Enriched virus-specific CD8+ T cells are analyzed by single-cell RNA sequencing, and single-cell heterogeneity is revealed

They analyzed the single-cell transcriptome and T cell receptor (TCR) sequence of >84,000 virus-reactive CD8+ T cells from 49 subjects in total, including patients with COVID-19 and healthy donors. Virus-reactive CD8+ T cells created seven clusters according to gene expression profiles, indicating heterogeneity among virus-reactive CD8+ T cells. They then described distinct characteristics of SARS-CoV-2-reactive CD8+ T cells compared to IAV-reactive or RSV-reactive CD8+ T cells. SARS-CoV-2-reactive CD8+ T cells from patients with COVID-19 and healthy donors were mainly composed of clusters enriched with T-cell exhaustion signature genes, IFN-stimulated genes, and cytotoxicity-related genes. In contrast, IAV-reactive or RSV-reactive CD8+ T cells were mainly composed of clusters enriched with inflammatory cytokine genes. They concluded that SARS-CoV-2-reactive CD8+ T cells exhibit exhausted phenotypes with type I IFN stimulation, and have a decreased capacity to secrete inflammatory cytokines.

Focusing on the transcriptome and TCR sequence data of SARS-CoV-2-reactive CD8+ T cells from patients with mild and severe COVID-19, they attempted to differentiate mild and severe COVID-19. SARS-CoV-2-reactive CD8+ T cells from patients with severe COVID-19 had a significantly lower frequency of the exhausted cluster than mild patients. When the analysis was narrowed down to the exhausted cluster, severe COVID-19-specific upregulated genes were highly enriched with cytotoxicity-related genes, pro-inflammatory cytokine genes, and genes for T-cell activation-associated transcription factors and negatively enriched with IFN response genes. These findings suggest that SARS-CoV-2-reactive CD8+ T cells are less exhausted, and more functional with an impaired type I IFN response in severe compared to mild COVID-19.

They also analyzed the non-exhausted cluster. Severe COVID-19-specific upregulated genes were enriched with genes related to co-stimulation and NF-κB activation, suggesting that SARS-CoV-2-reactive CD8+ T cells are more activated in patients with severe disease than those with mild disease. In the analysis of TCR clonality, clonal expansion was increased in SARS-CoV-2-reactive CD8+ T cells from patients with severe disease compared to those with mild disease. Collectively, SARS-CoV-2-reactive CD8+ T cells present a robust response in severe patients.

Kusnadi et al. reported a valuable resource for understanding the heterogeneity of the host immune response against SARS-CoV-2 infection by investigating SARS-CoV-2 reactive CD8+ T cells with the modified ARTE assay and scRNA-seq analysis. Unlike previous studies investigating total CD8+ T cells, this study described a landscape of SARS-CoV-2-reactive CD8+ T cells isolated by modified ARTE for the first time.

ARTE is a useful technique for enriching T cells reactive to specific antigens. However, it has inherent limitations for the proper characterization of antigen-reactive CD8+ T cells. Because the process for ARTE includes ex vivo stimulation of T cells with overlapping peptide antigens, the phenotypes and transcriptomes of antigen-reactive T cells can be changed by stimulation. In addition, ARTE cannot capture antigen-specific, non-functioning T cells. This is critical because a considerable proportion of antigen-specific CD8+ T cells detected by MHC-I multimer staining do not exert effector functions.5 These limitations can be overcome by using DNA barcode-tagged MHC-I multimers in scRNA-seq analysis (Fig. 1). MHC-I multimer staining enables the detection of virus-specific CD8+ T cells without stimulation regardless of their functions. However, MHC-I multimer combined scRNA-seq analysis has not yet been reported in the study of SARS-CoV-2-specific CD8+ T cells in patients with COVID-19.

One of the main findings by Kusnadi et al. is that SARS-CoV-2-reactive CD8+ T cells are mainly clustered in the exhausted subset. However, a recent study demonstrated that, among SARS-CoV-2-specific CD8+ T cells detected by MHC-I multimer staining, PD-1+ cells, as well as PD-1 cells, produced IFN-γ in patients with COVID-19 regardless of disease severity, indicating that SARS-CoV-2-specific CD8+ T cells are not exhausted, but functional.5 Further studies are required to examine the functional characteristics of CD8+ T cells in the exhausted cluster identified by Kusnadi et al. In addition, further studies are required to reveal a possible association between co-morbidities of COVID-19 patients and T cell functions.

The COVID-19 pandemic has urged us to investigate host immune responses, including the SARS-CoV-2-specific T-cell response. High-dimensional analysis adopting ARTE or MHC-I multimers will uncover the molecular characteristics, functions, and heterogeneity of SARS-CoV-2-specific CD8+ T cells in COVID-19 patients.


  1. 1.

    Kusnadi, A. et al. Severely ill COVID-19 patients display impaired exhaustion features in SARS-CoV-2-reactive CD8(+) T cells. Sci. Immunol. 6, eabe4782 (2021).

    Article  Google Scholar 

  2. 2.

    Mathew, D. et al. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science 369, eabc8511 (2020).

    CAS  Article  Google Scholar 

  3. 3.

    Lee, J. S. et al. Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19. Sci. Immunol. 5, eabd1554 (2020).

    CAS  Article  Google Scholar 

  4. 4.

    Grifoni, A. et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed Individuals. Cell 181, 1489–1501 (2020).

    CAS  Article  Google Scholar 

  5. 5.

    Rha, M. S. et al. PD-1-expressing SARS-CoV-2-specific CD8(+) T cells are not exhausted, but functional in patients with COVID-19. Immunity 54, 44–52 (2021).

    CAS  Article  Google Scholar 

Download references


This research was supported by the 2020 Joint Research Project of Institutes of Science and Technology.

Author information



Corresponding author

Correspondence to Eui-Cheol Shin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Koh, JY., Shin, EC. Landscapes of SARS-CoV-2-reactive CD8+ T cells: heterogeneity of host immune responses against SARS-CoV-2. Sig Transduct Target Ther 6, 146 (2021).

Download citation


Quick links