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. 2022 Apr 5;8:30. doi: 10.1038/s41421-022-00386-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Ratios (mean ± sd) of the 868 TCR mutants to the GAHDY*LN TCR in diploid yeasts induced in the YAMTAD screening. The NC peptides include TAX and 3*. Each experiment was performed with 4 technical repeats and was independently duplicated 2 times. The mean of 8 duplicates is shown. b The median MFI (mean ± sd) of the indicated 868 TCR mutants stained with PE fluorescently labeled SL9-HLA-A*02:01 tetramer. c A schematic diagram of the barcoding strategy used in library-on-library screening. A unique barcode was added to each TCR or pMHC strain. Expression of Cre recombinase induces recombination between the loxp 66 and loxp 71 sites, resulting in a juxtaposition of the MATa and MATalpha barcodes on the same chromosome. The two barcodes could be amplified by specific primers and subsequently sequenced simultaneously. d Ratios (mean ± sd) of diploids induced by the indicated peptide-TCR pairs to diploids induced by the negative control, the peptide-TAX 8* -TCR, in the YAMTAD library-on-library screening. The five peptides used for screening were mixed equally. Each experiment was performed with 4 technical repeats and was independently duplicated 3 times. The mean of 12 duplicates is shown. e Ratios (mean ± sd) of diploids induced by the indicated peptide-TCR pairs to diploids induced by the negative control, the peptide-3* -TCR, in the YAMTAD library-on-library screening. The SL9, 3 L, and TAX 8* peptides were mixed with the negative yeasts-3* and TAX at a 1:10² ratio. Each experiment was performed with at least 3 technical repeats and was independently duplicated twice. The mean of 7 replicates is shown. f Ratios (mean ± sd) of diploids induced by the indicated peptide-TCR pairs to diploids induced by the negative control, the peptide-3* -TCR, in the YAMTAD library-on-library screening. The SL9, 3 L, and TAX 8* peptides were mixed with the negative yeast- 3* and TAX at a 1:10⁴ ratio. Each experiment was performed with at least 3 technical repeats and was independently duplicated twice. The mean of 7 replicates is shown. Unpaired two-tailed Student’s t-test was used for statistical analysis in a, d, e, f. ** indicates a two-tailed P value < 0.01. * indicates a two-tailed P value < 0.05.