Fig. 7: CD103 overexpression deletes autoreactive 2D2 DN T cells in the periphery.
A Histograms identify T cells in the LNs of WT (n = 7), 2D2 (n = 6), 2D2.CD103Tg (n = 4) and 2D2.CD103Tgβ7Tg (n = 9) mice (left). Bar graphs show the frequency and number of LN T cells in the corresponding mice (right). Results are the summary of 3 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; ***, P < 0.001; NS, not significant. Frequency One-Way Anova P < 0.0001; Cell number One-Way Anova P = 0.0519. Data are presented as mean values ± SD. B Contour plots show CD4 versus CD8 profiles of LN T cells of WT (n = 7), 2D2 (n = 6), 2D2.CD103Tg (n = 4) and 2D2.CD103Tgβ7Tg (n = 9) mice (left). Bar graphs show the frequency and number of LN CD4+ T cells in the corresponding mice (right). Results are 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; NS, not significant. Frequency One-Way Anova P = 0.0003; Cell number One-Way Anova P < 0.0001. Data are presented as mean values ± SD. C Contour plots show CD62L versus CD44 expression in LN CD4 T cells of WT (n = 7), 2D2 (n = 6), 2D2.CD103Tg (n = 4), and 2D2.CD103Tgβ7Tg (n = 9) mice (left). Bar graphs (right) show the frequency and number of CD62Llo CD44hi memory phenotype CD4 T cells among LN CD4 T cells of the indicated mice. Results are the summary of 4 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; **, P < 0.01; NS, not significant. Frequency One-Way Anova P = 0.0042; Cell number One-Way Anova P = 0.1994. Data are presented as mean values ± SD. D Bar graphs show the frequency and number of LN DN T cells in WT (n = 7), 2D2 (n = 6), 2D2.CD103Tg (n = 4), and 2D2.CD103Tgβ7Tg (n = 9) mice (right). Results are the summary of 4 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant. Frequency One-Way Anova P < 0.0001; Cell number One-Way Anova P < 0.0001. Data are presented as mean values ± SD. E Contour plots (top) show intracellular IFN-γ contents in PMA/ionomycin-stimulated LN DN T cells of 2D2 (n = 6) and 2D2.CD103Tgβ7Tg (n = 9) mice. Bar graphs (bottom) show the frequency and absolute number of IFN-γ-expressing LN DN T cells in the indicated mice. Results show the summary of 4 independent experiments. Unpaired two-tailed Student’s t-test was used to calculate P values. **, P < 0.01; NS, not significant. Data are presented as mean values ± SD. F Contour plots show the recovery of in vitro activated donor CD4 T cells from Rag2–/– host mice, seven days after adoptive transfer (left). At the time of injection, donor T cells were comprised of 1:1 mixed WT (Thy1.1+) and 2D2 (Thy1.2+) or 1:1 mixed WT (Thy1.1+) and 2D2.CD103Tg (Thy1.2+) CD4 T cells. Bar graph shows the frequency of 2D2 or 2D2.CD103Tg CD4 T cells among total donor T cells (right). Results are the summary of a total of 5 adoptive transfer experiments of WT and 2D2 (n = 3) or WT and 2D2.CD103Tg (n = 2) mixed donor cells. Data are presented as mean values ± SD. G Histogram shows the CD103 expression on donor CD4 T cells of the indicated mice, seven days after adoptive transfer into Rag2-deficient host mice. H Clinical score of EAE-induced mice by adoptive transfer of in vitro activated 2D2 or 2D2.CD103Tg CD4 T cells. A total of 34 and 25 C57BL/6 mice were assessed for pathology upon injection of pathogenic 2D2 or 2D2.CD103Tg CD4 T cells, respectively. Two-Way ANOVA test was used to calculate the P value. **, P < 0.05. Data are presented as mean values ± SD. I Graph shows the cumulative disease incidence that was assessed for each adoptive transfer group in 2 independent EAE experiments. Open circles are the results from the first EAE experiment, and closed circles show the results from the repeat experiment.
