Fig. 7. Simultaneous MAPKi phenocopies Sestrin-deficiency in vivo.

(a) Phospho-flow analysis of sestrin2 expression in CD4⁺ T cells from 16-month old mice. (b) Erk, Jnk and p38 phosphorylation in CD4⁺ T cells from control Sesn1^+/- and Sesn1^-/- mice (20 months) vaccinated as in Figure 6. Data (n = 4) presented relative to those of control Sesn1^+/- mice, set as 1. (c) Correlation between reduced sestrin2⁺-p-Erk⁺/p-Jnk⁺/p-p38⁺ signaling and enhanced IFN-γ⁺ production in CD4⁺ T cells from Sesn1^-/- versus Sesn1^+/- mice after FLUAD vaccination (n = 4; r² = 0.82). (d) Splenocyte and (e) CD4⁺ T cell counts from 16-old mice injected with Erk (25 mg/kg), Jnk (16 mg/kg) and p38 (10 mg/kg) inhibitors either individually or together (triple MAPKi), vaccinated with FLUAD, and daily kept on MAPKi for five days. Results relative to those of DMSO-treated mice, set as 1. (f) MAPK-self phosphorylation in CD4⁺ T cells from mice as in (d-e) assessed by in vitro kinase assays, and presented relative to those of DMSO treated mice, set as 1. (g) FLUAD-driven IFN-γ production and IgG isotypic-switch among sestrin2⁺ CD4⁺ T and CD19⁺ B cells in mice as in (d-e), assessed by flow-cytometry. Pooled data (n = 4) presented relative to those of DMSO treated mice, set as 1. Data are pooled from four experiments with four (age-matched) mice per group (a, b, c, d, e, g) or two experiments (f). **p<0.01 and ***p< 0.001, ANOVA for repeated measures with Bonferroni post-test correction. Error bars indicate s.e.m. throughout. r², Pearson-correlation test.