Cd4 rm4 4

To avoid non-specific staining, Fc blocker (BD Pharmingen, USA) was applied before staining. Cells from LNs and spleens were incubated with antibodies against CD3e (145-2C11), CD19 (6D5), the TCR β chain (H131), TCR γδ (GL3), CD4 (RM4-4), and CD8a (53-6.7) (BD Pharmingen, USA). Thymocytes were incubated with antibodies against CD4 (RM4-4), CD8a (53-6.7), CD25 (PC61), CD44 (IM7), CD62L (MEL-17), and CD69 (H1.2F3) (BD Pharmingen, USA). Thymic stromal cells were incubated with antibodies against MHC class I (34-1-2S) and II (M5/114.15.2) and CD127 (A7R34) (Biolegend, USA). Intracellular Bcl-2 (BCL/10C4) staining of thymocytes was performed according to the manufacturer’s instructions provided with the kit (Biolegend, USA). FCM was performed on a Gallios (Beckman Coulter, USA) or Accuri C6 (BD, USA) flow cytometer.

N3tg/p50^−/− double-mutant mice at 3 weeks of age were injected intraperitoneally with 250 μg of InVivoPlus anti-mouse CD8α (2.43 clone), plus 250 μg of InVivoPlus anti-mouse CD4 (GK1.5 clone) or with 500 μg of InvivoPlus RatIgG2b (LTF-2 clone) isotype control (all from BioXCell), resuspended in 200 μl/mouse of PBS 1x, twice a week. After 3 weeks of treatment mice were sacrificed and characterized by FACS analysis, as described above. In particular, to evaluate the distribution of T cell subsets we used the following surface antibodies: CD4 (RM4-4, BD Bioscience) and CD8β (H35-17.2, eBioscience).