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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. 2 — a,b, EmbedSOM maps of concatenated DP thymocyte samples from Lck-variant mice. a, EmbedSOM maps show individual FlowSOM clusters and the relative expression of indicated markers. A representative experiment out of a total of five experiments is shown. b, Frequency of cells in cluster 5; n = 7 Lck^WT/WT, n = 4 Lck^KO/KO, n = 9 Lck^KR/KR, n = 8 Lck^CA/CA and n = 10 Lck^CA/KR in five independent experiments. Medians are shown. Statistical significance was calculated using a Mann–Whitney test. c, Thymocytes from indicated Lck-variant OT-I mice were activated with T2-Kb cells loaded with the indicated peptides (affinity: OVA > T4 > G4) and analyzed for CD69 expression by flow cytometry; n = 3 (OVA and T4) or 4 (G4) independent experiments/mice. d, Thymocytes of indicated Lck-variant B3K508 mice were activated with Ly5.1 splenocytes loaded with indicated peptides (affinity: 3K > P5R > P2A) and analyzed for CD69 expression by flow cytometry; n = 8 (3K and P2A in Lck^WT/WT and Lck^CA/CA mice), n = 7 (3K and P2A in Lck^CA/KR mice) or n = 5 (P5R) independent experiments/mice. Data in c and d are shown as mean + s.e.m. 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. The significance of the differences between Lck^WT/WT and Lck^CA/CA mice (blue) and Lck^WT/WT and Lck^CA/KR mice (red) at individual concentrations in d was calculated using a Mann–Whitney test; *P < 0.05; **P < 0.01; no symbol, P > 0.05 (Supplementary Table 3). e–g, TCR repertoires of FACS-sorted CD4⁺ and CD8⁺ mSP cells in the LNs and thymi from indicated mice were profiled. UMI-corrected counts of TCRα (TRA) and TCRβ (TRB) CDR3 amino acid sequences were normalized after the removal of NKT TRAV11-TRAJ18 CDR3 sequences. Sample sizes are in Supplementary Table 4. e, Principal-component analysis of all samples. f, Percentage of the repertoire in the indicated samples constituted by the top 20 most frequent CDR3 amino acid sequences in the LNs of Lck^WT/WT mice. g, The repertoire overlap was calculated as the percentage of unique CDR3 amino acid sequences in each sample present among the unique CDR3 sequences from each of the (non-identical) Lck^WT/WT mice. Each dot represents a single comparison.

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