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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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Extended Data Fig. 8 — (a-d) Thymi (a-b) and LNs (c-d) of indicated Lck variant F5 Rag1^KO/KO mice were analyzed by flow cytometry. (a) Expression of CD4 and CD8α in representative mice (gated on viable cells). (b) Counts of mature SP8 (viable CD4⁻ CD8α⁺ CD24⁻ TCRβ⁺) thymocytes. Individual mice and medians are shown. n = 10 Lck^WT/WT in 7 independent experiments, 5 Lck^KO/KO in 3 independent experiments, 9 Lck^KR/KR, 13 Lck^CA/CA and Lck^CA/KR in 8 independent experiments. (c) Expression of CD4 and CD8α in representative mice (gated on viable cells). (d) Total numbers of CD8⁺ T cells in LNs. Individual mice and medians are shown. n = 10 Lck^WT/WT in 7 independent experiments, 5 Lck^KO/KO in 3 independent experiments, 9 Lck^KR/KR, 12 Lck^CA/CA and 13 Lck^CA/KR in 8 independent experiments. (e-f) LN cells from indicated Lck variant OT-I mice were loaded with Cell Trace violet (CTV) and stimulated with anti-CD3/CD28 beads. (e) The proliferation was evaluated based on the CTV dilution at 72 hours after activation by flow cytometry. A representative experiment/mice out of 4 in total. (f) Upregulation of CD69 was analyzed by flow cytometry at 16 hours after activation. 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 extra sum-of-squares F test. F and p values are shown. (g) Thymocytes from indicated Lck variant CD8 WT or CD8.4 OT-I mice were analyzed by flow cytometry. Representative mice and the frequencies of SP4 T cells from 3 independent experiments/mice are shown. (h) LN cells from indicated Lck variant CD8 WT or CD8.4 OT-I mice were analyzed by flow cytometry. Representative mice and the frequencies of CD44⁺ CD49d⁻ antigen-inexperienced memory-like cells (individual values and means) from 3 independent experiments/mice are shown.

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