Figure 3.
Determination of epitopes of public Tfh clonotypes expanded in COVID-19 patients within conserved S regions exhibiting low mutation rates.(A) Characterization of public clonotypes. Clonotypes detected in multiple donors from our sample pool are indicated by # (column A). Clonotype occurrences were calculated based on a prepandemic healthy cohort from Japan (n = 27, column K) and cohorts of prepandemic healthy donors (n = 786) and convalescent COVID-19 patients (n = 1,413) from multiple ethnicities (column I). For clonotypes that contain different TCRα or TCRβ sequences, occurrences were calculated using pooled sequences (columns K and I). Clonotypes are listed following the order of significance of TCRβ expansion. Clonotypes expanded significantly in recovered patients are indicated by * (P < 0.05, column J). (B) The TCR pair of each clonotype listed in A was reconstituted into reporter cells, and their epitopes and restricting HLAs were identified. TCR expression levels of paired TCRs on reporter cells are indicated by surface CD3 expression as shown in the histograms. Filled histogram, TCR-reconstituted reporter cells; open histogram, parental cells. Semi-pooled peptide matrix described in Fig. S3 A was used for epitope determination, in which reporter cells were stimulated in the presence of HEK293T expressing shared HLAs or autologous APCs and GFP reporter activities are shown as heatmaps. Unless noted otherwise, a peptide name that starts with an S followed by a number in normal size refers to a 15-mer S peptide, and the number indicates the position of its N-terminal amino acid in S protein. Restricting HLAs were determined as described in Fig. S3, B–G; Table S2, and Materials and methods. Clonotypes 1, 6, and 8 did not respond to S peptide pools. (C) Locations of epitopes described in B are highlighted in red in the three-dimensional structure of the SARS-CoV-2 S protein (Protein Data Bank under accession no. 6XR8). Frequencies of mutation (mutation rate) defined relative to Wuhan-Hu-1 were calculated based on data from CoV-GLUE and GISAID. Data are representative of two independent experiments (B).

Determination of epitopes of public Tfh clonotypes expanded in COVID-19 patients within conserved S regions exhibiting low mutation rates. (A) Characterization of public clonotypes. Clonotypes detected in multiple donors from our sample pool are indicated by # (column A). Clonotype occurrences were calculated based on a prepandemic healthy cohort from Japan (n = 27, column K) and cohorts of prepandemic healthy donors (n = 786) and convalescent COVID-19 patients (n = 1,413) from multiple ethnicities (column I). For clonotypes that contain different TCRα or TCRβ sequences, occurrences were calculated using pooled sequences (columns K and I). Clonotypes are listed following the order of significance of TCRβ expansion. Clonotypes expanded significantly in recovered patients are indicated by * (P < 0.05, column J). (B) The TCR pair of each clonotype listed in A was reconstituted into reporter cells, and their epitopes and restricting HLAs were identified. TCR expression levels of paired TCRs on reporter cells are indicated by surface CD3 expression as shown in the histograms. Filled histogram, TCR-reconstituted reporter cells; open histogram, parental cells. Semi-pooled peptide matrix described in Fig. S3 A was used for epitope determination, in which reporter cells were stimulated in the presence of HEK293T expressing shared HLAs or autologous APCs and GFP reporter activities are shown as heatmaps. Unless noted otherwise, a peptide name that starts with an S followed by a number in normal size refers to a 15-mer S peptide, and the number indicates the position of its N-terminal amino acid in S protein. Restricting HLAs were determined as described in Fig. S3, B–G; Table S2, and Materials and methods. Clonotypes 1, 6, and 8 did not respond to S peptide pools. (C) Locations of epitopes described in B are highlighted in red in the three-dimensional structure of the SARS-CoV-2 S protein (Protein Data Bank under accession no. 6XR8). Frequencies of mutation (mutation rate) defined relative to Wuhan-Hu-1 were calculated based on data from CoV-GLUE and GISAID. Data are representative of two independent experiments (B).

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