Differentiation of Naive CD4+ T Cells

Naive CD4⁺ T cells were cultured in tissue culture–treated multiwell plates that were coated overnight with 2 μg/mL of anti-CD3ε (Bio X Cell) at 10⁶ cells/mL. Th1, Th2, and Th9 were cultured as previously described (59 (link)), and in some cases IL-6 and/or IL-21 was added to these cultures in varying concentrations as noted. In cytokine screening experiments, cells were cultured in flat-bottom, 96-well plates with plate-bound anti-CD3 and soluble anti-CD28 as per above. Cytokines were added at day 0 of culture (IL-4, 10 ng/mL; IL-6, 20 ng/mL; IL-12, 10 ng/mL; IL-21, 100 ng/mL; Pam3CSK4, 2 μg/mL; TGF-β, 2 ng/mL; TNF 100 ng/mL; see Supplemental Table 3 for other cytokines used), and cells were expanded 1:4 in fresh media with no additional cytokines day 3 of culture. All cytokines were purchased from PeproTech, except TGF-β, which was purchased from R&D Systems, Bio-Techne. Pam3CSK4 was purchased from InvivoGen.