Extended Data Fig. 6: Thymocytes expressing CD8-MP-skewed TCRs exhibit hallmarks of elevated TCR signaling.

a, Left: Representative flow cytometric analysis of CD73 expression by TCRβ⁺ CD8^SP thymocytes from a 7-week-old Rag2-GFP mouse. Right: Expression of Rag2-GFP on the CD73^– and CD73⁺ CD8^SP thymocyte populations. The percentage of cells falling in the indicated gates is denoted. Data are representative of two independent experiments. b, Representative flow-cytometric analysis of CD44 and CD122 expression by indicated CD73^– TCRβ⁺ Thy1.1⁺ CD8^SP CD8-MP TCRrg thymocytes 6 weeks after bone marrow reconstitution. The percentage of cells falling in the indicated gates is denoted. Data are representative of five independent experiments. c, Representative flow cytometric analysis of Ki67 expression by TCRβ⁺ CD8β⁺ Thy1.1⁺ cells from the thymi of TCRrg mice expressing the indicated CD8-MP TCRs (top) and CD8-Naive TCRs (bottom), analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of five independent experiments. d, Left: Summary plot of pooled data from (c) showing the frequency of TCRβ⁺ CD8β⁺ Thy1.1⁺ cells that are positive for Ki67 staining for the listed T cell clone. Right: Data from the left panel were pooled from the CD8-MP TCRs and the CD8-Naive TCRs. Each symbol represents an individual TCRrg mouse. n = 11, CD8-MP TCRrg mice; n = 10, CD8-Naive TCRrg mice. Mean ± SEM is indicated. **p = 0.0017, two-tailed nonparametric Mann–Whitney test. Data are pooled from five independent experiments. e, Representative flow cytometric analysis of CD5 expression by TCRβ⁺ CD8β⁺ Thy1.1⁺ cells from the thymi of TCRrg mice expressing the indicated CD8-MP and CD8-Naive TCRs, analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of six independent experiments. f, Left: Summary plot of pooled data from (e) showing the normalized mean florescence intensity (MFI) of CD5 in TCRrg thymocytes compared to CD8^SP thymocytes from a B6 thymus. Right: Data from the left panel were pooled from the CD8-MP TCRs and the CD8-Naive TCRs. Each symbol represents an individual TCRrg mouse. n = 13, CD8-MP TCRrg mice; n = 12, CD8-Naive TCRrg mice. Mean ± SEM is indicated. ***p < 0.0001, two-tailed nonparametric Mann–Whitney test. Data are pooled from six independent experiments. g, Representative flow cytometric analysis of CD4 vs. CD8, PD-1 vs. CD69, and cleaved Caspase3 expression by TCRβ⁺ CD8β⁺ Thy1.1⁺ cells from the thymi of TCRrg mice expressing the indicated CD8-MP TCRs and CD8-Naive TCRs, analyzed 6 weeks after bone marrow reconstitution. The percentage of cells in the indicated gates is denoted. Data are representative of three independent experiments.