Fig 1: Escape of two naturally occurring SARS-CoV-2 mutations from S RBM-specific CD8+ T cells(A and B) Detection of HLA-A24-restricted NF9-specific CTLs. HLA-A24-positive CTL lines of 3 seronegative donors and 16 COVID-19 convalescent donors as well as 6 HLA-A24-negative COVID-19 convalescent donors (listed in Table S2) were stimulated with or without 1 µM NF9 peptide (NYNYLYRLF, residues 448–456 of the SARS-CoV-2 S protein). (A) Representative FACS plots showing surface expression of CD25 and CD137 on the CD8+ T cell subset (i.e., CD3+CD8+ cells) of an HLA-A24-positive seronegative donor (left), an HLA-A24-negative COVID-19 convalescent donor (middle), and an HLA-A24-positive COVID-19 convalescent donor #1 (right). (B) The median of the percentage of CD25+CD137+ cells among CD8+ T cells.(C and D) Multifunctionality of HLA-A24-restricted NF9-specific CTLs. HLA-A24-positive CTL lines of eight COVID-19 convalescent patients were stimulated with or without 10 nM NF9 peptide. Representative FACS plots showing intracellular expression of IFN-?, TNF-a, and IL-2 in the CD8+ T cell subset of COVID-19 convalescent donor #1 (C) and pie charts showing the proportion of cytokine-positive cells in each convalescent sample (D) are shown.(E and F) Potential killing activity of HLA-A24-restricted NF9-specific CTLs. HLA-A24-positive CTL lines of eight COVID-19 convalescent donors were stimulated with C1R-A2402 cells pulsed with or without 10 nM NF9 peptide. Representative FACS plots showing surface expression of CD107a in the CD8+ T cell subset of a COVID-19 convalescent donor #1 (E) and the median of the percentage of CD107a+ cells in CD8+ T cells (F) are shown.(G) Distribution of the L452R and Y453F mutants during the pandemic. Shown are the top five countries where variants harboring the L452R (top) and Y453F (bottom) mutations are found. The raw data are summarized in Table S3.(H and I) Mutations conferring escape from HLA-A24-restricted NF9-specific CTLs. HLA-A24-positive CTL lines of eight COVID-19 convalescent donors were stimulated with 1 nM NF9 peptide or its derivatives: NF9-L452R (NYNYRYRLF) and NF9-Y453F (NYNYLFRLF). Representative FACS plots showing intracellular expression of IFN-? in the CD8+ T cell subset of a COVID-19 convalescent donor #1 (H) and the mean percentage of IFN-?+ cells in CD8+ T cells (I) are shown.In (A, E, and H), the numbers in the FACS plot represent the percentage of gated cells among CD8+ T cells. In (C), the number represents the percentage of cells in each quadrant.In (B), a statistically significant difference (*p < 0.05) is determined by the Wilcoxon signed-rank test. NS, no statistical significance.In (F), each symbol of the COVID-19 convalescent data represents the mean of technical triplicates. Statistically significant differences (*p < 0.05) between COVID-19 convalescent samples with and without the NF9 peptide are determined by the Wilcoxon signed-rank test.In (I), the assay was performed in triplicate, and the means are shown with the SD. Statistically significant differences (*p < 0.05) versus “no peptide” are determined by ANOVA, with multiple comparisons by Bonferroni correction.See also Figure S1 and Tables S1, S2, and S3.