In a recent study published in Science Immunology, 1 Kusnadi et al. performed single-cell RNA sequencing (scRNA-seq) of SARS-CoV-2reactive 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 . During viral infection, CD8 + T cells contribute to elimination of the virus

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... exerting cytotoxicity against virusinfected 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-2reactive CD8 + and CD4 + T cells using ex vivo antigen stimulationbased 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-2specific 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 proteinreactive CD8 + T-cell population. 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, proinflammatory cytokine genes, and genes for T-cell activationassociated 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