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. 2021 Dec 6;40(4):488–498. doi: 10.1038/s41587-021-01089-x

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© The Author(s) 2021

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. 2 — a, Histograms of viable CD8⁺ T cells transduced with T1 and T3 TCRs stained with anti-mouse TCR-β antibody or relevant peptide–MHC multimers. b, Activation of T1 and T3 cells following coincubation with peptide-pulsed T2 lymphoblast cells. Data are pooled from three independent experiments, with each circle representing the mean of three technical replicates in each experiment. Data are shown as mean ± s.d. c, CD137 upregulation on CD8⁺ T1 (green) and T3 (purple) cells after coculture with EBV-LCLs derived from one HLA-A2^pos and one HLA-A2^neg donor, either pulsed with relevant peptides or electroporated with mRNA encoding full-length TdT. d, Activation of CD8⁺ T1 and T3 cells after coculture with TdT^pos cell lines REH (B-ALL) and HPB-ALL (T-ALL) in the presence/absence of introduced expression of HLA-A2, or pan-MHC class I blocking antibody W6/32 (Ab). e, IFN-γ production by T1 and T3 cells following coincubation with TdT^pos NALM-6 cells (WT), or NALM-6 cells in which TdT_475-481 was deleted by CRISPR–Cas9 (KO) in the presence/absence of added peptide. f, Activation of T1 and T3 cells after coculture with various cell lines, with indicated HLA-A2 and TdT expression, loaded or not with TdT peptides (2 × 10⁻⁷ M). The suffix + A2 denotes cell lines transduced with HLA-A*02:01. c–f, Results are from one experiment representative of two or three performed with different T-cell donors. Bars or connecting lines show mean, and individual data points represent either two (e) or three (c,d,f) technical replicates. g, Heat maps of IFN-γ produced by T1 (green) and T3 cells (purple) coincubated with EBV-LCLs pulsed with peptides from mimotope libraries. White circles, amino acid in wild-type peptide. IFN-γ concentration range for positive reactions was 500–31,254 pg ml^–1. One replicate per condition (see Extended Data Fig. 2c for correlation with CD137 activation assay). h, Model structures of TdT peptide-1 (green) and -3 (purple) represented as sticks, bound to the HLA-A2 molecule, in gray, shown from top (left) and side views (right). Individual amino acids are labeled with positional number and symbol.

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