Fig. 3: CD103 overexpression promotes negative selection in 2D2 transgenic mice.

A Contour plots show the CXCR4 versus CCR7 profiles of WT (n = 8), 2D2 (n = 6), and 2D2.CD103^Tgβ7^Tg (n = 9) thymocytes, identifying three distinct populations based on their CCR7 levels, i.e., CCR^neg, CCR7^int, and CCR7^hi. Bar graphs (right) show the frequencies of CCR7^int thymocytes (boxed in red, left) of the corresponding mice. Contour plots are representative, and the bar graph shows the summary of 4 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05, ***, P < 0.001. One-Way Anova P < 0.0001. Data are presented as mean values ± SD. B Contour plots (top) show CD4 versus CD8 profiles of CCR7^hi and CCR7^int thymocytes of 2D2 mice. Histogram (bottom) shows CD69 expression on DN (black) and DP (red) cells of CCR7^int-gated 2D2 thymocytes. Contour plots are representative of 4 independent experiments. C Contour plots show the CD69 versus CCR7 profiles of WT (n = 8), 2D2 (n = 6), and 2D2.CD103^Tgβ7^Tg (n = 9) thymocytes that identify six distinct populations based on their differential expression of CD69 and CCR7. Starting from CD69, CCR7 double-negative cells that we refer to as population I, six populations are identified in a clockwise manner, whereby population VI (CD69-negative, CCR7^int) is marked in red (left). Contour plots are representative of 4 independent experiments. Bar graph shows the frequency of population VI cells of the indicated mice. Data are a summary of 3 independent experiments. One-Way Anova P < 0.0001. Unpaired two-tailed Student’s t-test was used to calculate P values. **, P < 0.01, and ***, P < 0.001. Data are presented as mean values ± SD. D Contour plots show the CD4 versus CD8 profiles of population V (top) and VI (bottom) from 2D2 and 2D2.CD103^Tgβ7^Tg mice. Results are representative of 3 independent experiments. E Histograms show surface CD103 (top) and nuclear Helios expression (bottom) of population V CD4 T cells and population VI DN cells of 2D2 thymocytes. Results are representative of 3 independent experiments. F Histograms show PD-1 expression on population V CD4 T cells and population VI DN cells of 2D2 and 2D2.CD103^Tgβ7^Tg thymocytes. Results are representative of 3 independent experiments. G Histograms show the intracellular staining for active caspase-3 in population V CD4 T cells and population VI DN cells of 2D2 thymocytes. Bar graphs are a summary of 3 independent experiments with 2D2 mice (n = 5). Unpaired two-tailed Student’s t-test was used to calculate P values. ***, P < 0.001. Data are presented as mean values ± SD. H The frequencies of population VI cells were assessed among CXCR4-negative DN thymocytes of WT and CD103-deficient (Itgae^–/–) mice. Contour plots are representative and bar graph is a summary of 4 independent experiments with a total of 6 Itgae^–/– mice. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05. Data are presented as mean values ± SD.