In Vitro Th2 Cell Differentiation

Splenic naive T helper cells were purified with the CD4⁺CD62L⁺T Cell Isolation Kit II (Miltenyi Biotec) and polarized in vitro toward differentiated Th2 subtype as described before in [32 (link)]. In brief, naive cells were seeded into anti-CD3e (2 μg/ml, clone 145-2C11, eBioscience) and anti-CD28 (5 μg/ml, clone 37.51, eBioscience) antibody coated 96-well round bottom plates. The medium contained the following cytokines and/or antibodies for Th2 subtype: recombinant murine IL-2 (10 ng/ml, R&D Systems), recombinant murine IL-4 (10 ng/ml, R&D Systems), and neutralizing anti-IFN-g (5 μg/ml, cloneXMG1.2eBioscience). The cells were removed from the activation plate on day 4 (72 h). Th2 cells were cultured for another 2 days in the absence of anti-CD3 and CD28 stimulation. Then, cells were restimulated by anti-CD3e/CD28-coated plate for 6 h. For flow cytometric detection, cells were treated with monensin (2 μM, eBioscience) for the last 3 h.