In vitro T cell differentiation assays

Human CD4⁺ T cells and mice CD4⁺ T cells were purified using negative selection (CD4⁺ T Cell Isolation Kit, human; CD4⁺ T cell Isolation kit, mouse; Miltenyi Biotec, Auburn, CA) respectively. Purified human and mice CD4⁺ T cells were stimulated in the presence of anti-CD3 (5 μg/ml, UCHT1; 1 μg/ml, 145-2C11) and anti-CD28 (1 μg/ml, CD28.2; 1 μg/ml, 37.51) (BD Bio-sciences) for 3d under the following conditions—For human CD4⁺ T cells: Th1: rhIL-12 (10 ng/ml; Peprotech, Rocky Hill, NJ); Th2: rhIL-4 (25 ng/ml; Peprotech); Th17: TGF-β (5 ng/ml; Peprotech), IL-1β, IL-21 and IL-23 (all 25 ng/ml; Peprotech); Treg: 1 ng/ml rhTGF-β and 2 ng/ml rhIL-2 (Peprotech). Recombinant human IL-2 was bought through ACRO Biosystems and was used at 10 U/ml, except under Treg (2 ng/ml) and Th17 conditions. For mice CD4⁺ T cells: Th1: 40 ng/ml rIL-12 (Peprotech) and 40 μg/ml anti-IL-4 (R & D); Th2: 40 ng/ml rIL-4 (Peprotech) and 10 μg/ml anti-IFN-γ (R & D); Th17: 1 ng/ml rTGF-β and 40 ng/ml rIL-6 (both Peprotech), 40 μg/ml anti-IL-4 and 10 μg/ml anti-IFN-γ (both R & D); Treg: 1 ng/ml rTGF-β and 2 ng/ml rIL-2 (both Peprotech).