Figure 7. IKAROS GOF enhances Th2 polarization through the FYN/ITCH/JunB axis.

(A) Immunoblot analysis of JunB in T cell blasts derived from healthy controls (n = 3) and the patients (n = 3) after anti-CD3/anti-CD28 stimulation for 24 hours. (B) Immunoblot analysis of JunB in T cells from healthy individuals; the cells were lentivirally transduced with empty vector or an FYN-encoding vector. JunD and c-Jun were stained as a control to rule out nonselectivity. (C) Immunoprecipitation and immunoblot analysis of ubiquitinated (Ub) JunB in T cell blasts derived from a healthy control and a patient after anti-CD3/anti-CD28 stimulation for 6 hours. Representative blots and summary of 3 experimental replicates are shown. (D) Immunoblot analysis of FYN and JunB in naive CD4⁺ T cells from healthy individuals; the cells were lentivirally transduced with WT IKFZ1 or R183H variant–encoding vectors. (E and F) WT IKFZ1 and R183H (RH) lentivirally transduced naive CD4⁺ T cells from healthy individuals were transfected with control or FYN siRNA (E) or control or JUNB siRNA (F). Cells were cultured on anti-CD3/anti-CD28–coated plates for 7 days. mRNA expression of TBX21 and GATA3 was determined by qPCR after PMA and ionomycin treatment for 2 hours. Data represent the mean ± SEM (E and F). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, unpaired Student’s t test (A), 2-tailed, paired Student’s t test (B–D), or 2-way ANOVA (E and F).