ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 6

Figure 6. Concurrent deletion of MDA5 rescues impaired thymic T cell maturation in CD4+ cell-specific Adar1 knockout mice.

A CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with MDA5 knockout (Ifih1-/-) mice to generate Cd4cre A1fl/fl Ifih1-/- mice. Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The percentage of DN, DP, 4SP and 8SP thymocytes were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl (n = 7) mice (right panel). The same data shown in Fig 2C was used for Cd4cre A1fl/fl mice. Values are displayed as the mean ± SEM (Mann-Whitney U-test, *p < 0.05, **p < 0.01, N.S.; not significant).

B The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in Ifih1-/- 4SP thymocytes was set as one.

C Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, N.S.; not significant) (right panel) by setting the mean value of stimulated Ifih1-/- samples at 60 sec as 1.

D Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.

E Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared among Ifih1-/- (n = 5), Cd4cre A1fl/fl Ifih1-/- (n = 3) and Cd4cre A1fl/fl (n = 3) mice (lower panel). The same data shown in Fig 2F was used for Cd4cre A1fl/fl mice. Values are displayed as the mean ± SEM (Mann-Whitney U-test, *p < 0.05, N.S.; not significant).

F Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared among Ifih1-/-, Cd4cre A1fl/fl Ifih1-/- and Cd4cre A1fl/fl mice (lower panel). The same data shown in Fig 2H was used for Cd4cre A1fl/fl mice. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05, N.S.; not significant).