Extended Data Fig. 2: Phenotype of OTI TCR transgenic Themis cKO mice.

(A) Anti-Themis Western blotting of cell lysates from FACS-sorted DP and CD8 SP thymocytes,with anti-tubulin as aloading control. Data representative of 2 independent experiments. (B) Percentages of CD8 SP thymocytes, CD69^low CD8 SP thymocytes and TCR^hiCD24^low CD8 SP thymocytes from OTI WT and cKO mice. Mean +/- SEM from 13 mice/genotype, from 4 independent experiments. (C) (left) CD5 MFI on CD8+ T cells from Themis cKO ERT2-Cre+ and Cre-mice control mice after tamoxifen treatment. Mean +/- SEM from 12 mice/genotype from 4 independent experiments. (right) CD5 MFI as percentage of ERT2-Cre- mean CD5 MFI. Mean +/- SEM from 21 mice from 7 independent experiments. (D) CD8 and TCRβ MFI on OTI cKO CD8 SP thymocytes and CD8⁺ lymphocytes as percentage of WT mean MFI. Mean +/- SEM from 13-17 mice/genotype from 4-5 independent experiments. (E) Ex vivo Eomes nuclear staining. SEM +/- SEM from data from 7-8 mice from 3 independent experiments. (F) Percentages of CD44^lowCD62L^hi, CD44^hiCD62^low, Cd44^hiCD62L^hi and CD44^lowCD62^low populations. Mean +/- SEM from 13 mice/experiment, from 4 independent experiments. (G) Increase in Va2^low CD8+ T lymphocytes and CD8 SP thymocytes in Themis cKO OTI mice. The flow cytometry plots representative of 13 mice/genotype from 4 independent experiments, and the numbers shown indicate mean +/- SEM of 13 mice/genotype from 4 independent experiments. Graphs show percentages of the indicated populations from 13 mice/genotype from 4 independent experiments (Rag1 WT) or 10 mice/genotype from 2 independent experiments (Rag1 KO). Graph showing CD5 MFI on Vα2^low cells as percentage of CD5 MFI on Vα2^hi cells shows data from 11 mice/genotype from 3 independent experiments. WT and cKO samples were compared using unpaired t-test, with *** indicating p-values from 0.0001 to 0.001, ** indicating p-values from 0.001 to 0.01. Mean +/- SEM values are shown.

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