Effect of ASB2 deletion on gene expression in non-stimulated and stimulated mouse Th2 lymphocytes
Because ASB2a-mediated degradation of filamin A and filamin B could be involved in the modulation of Th2 lymphocyte signaling and thus in the regulation of Th2-specific genes, the transcriptomic signatures of ctrl and ASB2a-deficient Th2 lymphocytes were established by RNA-seq. We here report no impact of ASB2a loss and filamin A/filamin B accumulation on the transcriptomic program of Th2 lymphocytes. Overall design: Ctrl and ASB2 cKO mouse naive CD4+ cells were isolated from spleen of female Asb2fl/fl and VE-cadherin (VEC) -Cre;Asb2fl/fl transgenic mice (Metais et al., Circulation Research, 2018; Lamsoul et al., Blood, 2013), respectively, using a CD4+ T-cell Isolation Kit (BioLegend) and cultured for 3 days with plate-bound anti-CD3e (5 µg/ml) in the presence of Th2 polarizing cytokines and antibody cocktails (10 ng/ml IL-2, 40 ng/ml IL-4, 1 µg/ml anti-CD28, and 10 µg/ml anti-IFN?) in RPMI containing 10% FCS, 1% glutamine, 0.1% ß-mercaptoethanol, and 1% penicillin/streptomycin in 24-well plates. Cells were then split across five noncoated plates and cultured for 3 additional days in fresh differentiating medium without antibodies (20 ng/ml IL-2 and 20 ng/ml IL-4). Th2 lymphocytes were stimulated at day 5 with 3µg/ml coated anti-CD3e and 2µg/ml soluble anti-CD28 for 24h. RNA isolation was performed using the Nuclespin RNA kit (Macherey-Nagel).