TRAF6-deficient CD4⁺ T cells exhibit increased Th17 differentiation in vitro. (A) CD4⁺ T cells isolated from TRAF6-ΔT and control mouse spleens were activated with anti-CD3 and anti-CD28 with accessory cells in Th1, Th2, and Th17 conditions. Intracellular cytokine content was determined by flow cytometry after 4-day culture. Cells were cultured in triplicate, and the experiment was repeated with similar results. (B) Relative RNA levels of transcription factors t-bet, GATA-3, and RORγt were determined by real-time PCR analysis. The experiment was repeated with similar results. (C) Naive (CD62L⁺CD44^loCD25⁻) CD4⁺ T cells isolated from lymph nodes and spleen were stimulated with plate-coated anti-CD3 and soluble anti-CD28 under optimal Th17 conditions. Intracellular cytokine content was determined by flow cytometry after 5-day stimulation. Right panel shows results from 4 independent experiments. *P < .05.