Mouse T cell cultures

CD4⁺ CD25^- T cells were isolated ex vivo from spleens and lymph nodes of mice by enrichment with EasyStep™ Mouse CD4⁺ Isolation Kit (Stemcell Technologies) in combination with a biotin-conjugated antibody against CD25, followed by immunomagnetic negative isolation. The purity of the isolated cells was ∼90%. RPMI 1640 GlutaMAX™ medium or IMDM GlutaMAX™ medium (both from Life Technologies) was used for Th1, Th2, and Treg or Th17 cell cultures, respectively. The medium was supplemented with 10% heat-inactivated FCS (Biochrom), 500 U penicillin-streptomycin (PAA laboratories), and 50 mM β-mercaptoethanol (Life Technologies). For Th17 cell induction, 1.5 to 3×10⁵ naive T cells were cultured for 4 days with plate-bound aCD3ε (10 mg mL^–1, clone 145-2C11; Bio X Cell), aCD28 (1 mg mL^–1, clone 37.51; Bio X Cell), aIFN-g (5 mg mL^–1, clone XMG1.2; Bio X Cell), aIL-4 (5 mg mL^–1, clone 11B11; Bio X Cell), rhTGF-β1 (2 ng mL^–1; Peprotech), rmIL-6 (7.5 ng mL^–1; Peprotech), and rmIL-1b (50 ng mL^–1; Peprotech). For Th1 induction, 1×10⁵ naive T cells were cultured for 4 days in the presence of plate-bound aCD3ε (10 mg mL^–1), aCD28 (1 mg mL^–1), rmIL-12 (50 ng mL^-1), and aIL-4 (5 mg mL^–1). For Th2 induction 1×10⁵ naive T cells were cultured for 4 days with plate-bound aCD3ε (10 mg mL^–1, clone 145-2C11; Bio X Cell), and aCD28 (10 mg mL^–1, clone 37.51; Bio X Cell), aIFN-γ (10 mg mL^–1, clone XMG1.2; Bio X Cell), aIL-12 (10 mg mL^–1, clone 17.8; Bio X Cell), and rmIL-4 (1 mg mL^–1, Preprotech). Arg C, Arg F, Linezolid (Chem-Impex), Tigecycline hydrate (Sigma), or Thiamphenicol (Chem-Impex) were added at the indicated concentrations at the onset of the culture. For proliferation analysis, naive T cells were labeled using 5μM CellTrace™ Violet Cell Proliferation Kit (Life Technologies) after magnetic sorting.