Fig. 1: Cellular screening of SARS-CoV-2-specific response.

a Memory CD8⁺ T cells (CD45RO⁺) from peripheral blood mononuclear cells (PBMC) of COVID-19 convalescents were seeded at 2000 cells/well in multiple wells containing irradiated allogenic PBMC, PHA, IL-2, IL-7, and IL-15 as described in Methods. The individual cultures were expanded to make up the libraries of polyclonally amplified CD8⁺ T cells. Then the libraries were washed and examined as to their antigen specificity by stimulating with a series of viral protein-expressing aAPC. Once being judged as positive by IFNγlevel (measured by ELISA), they were further expanded with relevant aAPC and cytokines. These libraries (cell lines) were subjected to several downstream analyses including epitope screening, cytokine profiling, and/or cytotoxicity assays, etc. b A representative result of the CD8⁺ T cell library assay. Libraries from a healthy donor (HC-010) or COVID-19 convalescent (CV-004) were divided into 7 groups, and each group was co-cultured with indicated aAPCs: S (n = 15 for HC-010; n = 47 for CV-004), M (n = 15 for HC-010; n = 47 for CV-004), N (n = 15 for HC-010; n = 47 for CV-004), ORF3a (n = 14 for HC-010; n = 41 for CV-004), or ORF1ab NSP6 (n = 14 for HC-010; n = 53 for CV-004) -expressing aAPCs, influenza virus and cytomegalovirus (Flu/CMV) derived peptides (n = 15 for HC-010; n = 47 for CV-004)-pulsed aAPC or aAPCs (None) (n = 15 for HC-010; n = 47 for CV-004). Each dot represents the IFNγ level of each library. The threshold of positivity was determined by mean + 3 SD of IFNγ level in the group co-cultures with aAPCs (indicated as “None”). c Heatmap representation of results from all participants (healthy controls (HC), convalescents from mild, moderate (mod), and severe (sev) disease) subjected to the library assay. The frequency of positive library in response to aAPCs (None), indicated viral protein-expressing, or influenza virus and cytomegalovirus (Flu/CMV) derived peptides-pulsed aAPC are shown. d Comparison of positive library frequency between healthy donors (HC) (open circles, n = 8) and COVID-19 convalescents (COVID-19) (filled circles, n = 20) in each group. p values were calculated by two-sided Mann–Whitney test. Data represent mean ±  SD. **p = 0.0064. e Comparison of positive library frequency in response to SARS-CoV-2-M protein-expressing aAPC among indicated groups (healthy donors (HC) (n = 8), convalescents from mild severity COVID-19 (mild COVID-19) (n = 8), those from moderate severity COVID-19 (moderate COVID-19) (n = 8) and those from severe severity COVID-19 (severe COVID-19) (n = 4)). p values were calculated by two-sided Mann–Whitney test. Data represent mean ± SD. ***p = 0.0002; *p = 0.024.