Moflow

Lymphocytes were isolated from spleen and peripheral lymph nodes (including inguinal, auxiliary and cervical lymph nodes) or thymus. Naïve CD4⁺ T cells (CD4⁺CD25⁻CD44⁻CD62L⁺), Treg cells (CD4⁺YFP⁺ or CD4⁺CD25⁺ as indicated in the figures and text) and thymic mature CD4⁺ T cells (CD4⁺CD8⁻CD69^loCD62L^hi) were sorted on a MoFlow (Beckman-Coulter) or Reflection (i-Cyt). Sorted cells were cultured in plates coated with anti-CD3 (2C11, 10 μg/ml) and anti-CD28 (37.51, 10 μg/ml) for 4.5 days in Click's medium (Irvine Scientific) supplemented with β-mercaptoethanol, 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin and IL-2 (200 U/ml). In certain experiments, Treg cells were stimulated with IL-2 (0-500 U/ml) as indicated in the figures and legends. CD62LhiCD69lo mature thymocytes from WT and Cd4^CreStk4^f/fStk3^f/f mice were differentiated under induced Treg skewing condition (2 μg/ml CD3, 2 μg/ml CD28, 1 ng/ml TGF-β, and 100 U/ml IL-2) with irradiated antigen-presenting cells for 5 days, and Foxp3 expression was examined by flow cytometry. For activated CD4⁺ T cell blast generation, peripheral CD4⁺CD25⁻CD44⁻CD62L⁺ naïve T cells were sorted and stimulated with plate-bound 10 μg/ml CD3⁺CD28 for 3 days and rested in 100 U/ml IL-2 for another 3 days. Live T cells were purified using Ficoll, and rested without IL-2 for 8 h and then stimulated with IL-2 for the indicated time points.