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. 2022 Dec 23;24(1):174–185. doi: 10.1038/s41590-022-01366-0

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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–d, Thymi (a and b) and LNs (c and d) of indicated Lck-variant OT-I mice were analyzed by flow cytometry. a, Expression of CD4 and CD8α in representative mice (gated on viable cells). b, Numbers of mSP8 (viable CD4⁻CD8α⁺CD24⁻TCRβ⁺) T cells. Individual mice and medians are shown; n = 22 Lck^WT/WT mice in 12 independent experiments, n = 12 Lck^KO/KO mice in 8 independent experiments, n = 14 Lck^KR/KR mice in 6 independent experiments, n = 13 Lck^CA/CA mice in 7 independent experiments and n = 11 Lck^CA/KR mice in 3 independent experiments. The statistical significance was tested using a Mann–Whitney test. c, CD4⁺ and CD8⁺ T cells in representative mice (gated on viable TCRβ⁺ cells). d, Counts of CD8⁺ T cells. Individual mice and medians are shown; n = 23 Lck^WT/WT mice in 12 independent experiments, n = 12 Lck^KO/KO mice in 8 independent experiments, n = 14 Lck^KR/KR mice in 6 independent experiments, n = 13 Lck^CA/CA mice in 7 independent experiments and n = 11 Lck^CA/KR mice in 3 independent experiments. Statistical significance was tested using a Mann–Whitney test. e, T cells isolated from LNs of indicated Lck-variant OT-I Rag2^–/– mice were activated ex vivo with T2-Kb cells loaded with the indicated peptides (affinity: OVA > Q4R7 > T4 > G4 > E4 > E1) overnight and analyzed for expression of CD69 by flow cytometry. Data are shown as the mean + s.e.m.; n = 3 independent experiments/mice for OVA and Q4R7 and n = 4 independent experiments/mice for T4, G4, E4 and E1. Differences in the EC₅₀ and/or maximum of the fitted non-linear regression curves were tested using an extra sum of squares F-test. F, P and EC₅₀ values are shown. f, Jurkat cells expressing OT-I TCR and LCK^WT were transduced with CD8^WT or with CD8^CA. These Jurkat cells were activated with T2-Kb cells loaded with OVA peptide overnight and analyzed for CD69 expression by flow cytometry. Data are shown as mean + s.e.m.; n = 3 independent experiments/mice. Differences in the EC₅₀ and/or maximum of the fitted non-linear regression curves were tested using an extra sum of squares F-test, and P, F and EC₅₀ values are shown.

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