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. 2021 Aug 19;218(10):e20210785. doi: 10.1084/jem.20210785

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© 2021 Greaves et al.

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Figure 2. — Deconvolution strategy to define peptides that stimulate LS-specific TCRs. (A) IL-2 responses of LS-specific 1244c1 (red bars) and 1244c3 (blue bars) CD4⁺ T cell hybridomas after stimulation with a decapeptide PSL presented by a fibroblast cell line (B6DK10) expressing HLA-DR3 after a 24-h incubation. In each mixture, one position is fixed with each of the 20 amino acids, which is denoted on the x axis with the single-letter code. Unfixed positions in each decapeptide mixture consist of an equimolar mixture of 19 amino acids (cysteine is excluded). Peptide mixtures were tested at 200 μg/ml, and IL-2 responses were measure by ELISA. Data are representative of two independent experiments. (B) LS-specific 1244c1, 1244c2, and 1244c3 CD4⁺ T cell hybridomas were stimulated with 37 dual-defined PSL mixtures at 100 μg/ml. Fixed amino acids and their positions in each decapeptide mixture are labeled. Unfixed positions in each decapeptide mixture consist of an equimolar mixture of 19 amino acids (cysteine is excluded). Dual-defined doublet mixtures are included to test register shifting in the unbiased PSL results. In the remaining dual-defined mixtures, two different TCR peptide recognition registers are apparent, denoted as register 1 and register 2. IL-2 responses of the LS-specific 1244c1 (red), 1244c2 (black), and 1244c3 (blue) CD4⁺ hybridomas stimulated with a dual-defined PSL are shown. Data are representative of three independent experiments. (C) Sequence logos summarizing LS-specific 1244c1 (top), 1244c2 (middle), and 1244c3 (bottom) CD4⁺ hybridoma responses to the G2D4N5 PSL are shown. The x axis denotes the position of the decapeptide, and the y axis denotes the relative IL-2 activity of each TCR when stimulated with peptide mixtures at 200 μg/ml. The color of each amino acid represents its chemical property: green, polar; purple, neutral; blue, basic; red, acidic; and black, hydrophobic. (D) Selection of amino acids for each peptide position for the 1244 LS-specific TCRs based on the most active mixtures from the unbiased PSL and G2D4N5 PSL.