Naïve CD4+ T cells were either left unstimulated or stimulated by cross-linking the CD3 receptor (simulating TCR ligation), with flagellin (to activate TLR5), with both stimuli (TCR + TLR5 activation), or by cross-linking CD3 and CD28 (TCR + CD28 activation). The activation of the following molecules was evaluated by flow cytometry: p65 (pp65), CREB (pCREB), and c-Jun (pc-Jun). Cells were stimulated in RPMI medium supplemented with 2% FBS. The following antibodies were used at 1 and 0.1 μg/ml in different combinations: anti-CD3 (OKT3) antibody (Tonbo), anti-CD28 (CD28.2) antibody (Tonbo), and polyclonal goat anti-mouse antibody (BioLegend) to cross-link the anti-CD3 and anti-CD28 antibodies. TLR5 signals were induced with flagellin monomer (InvivoGen) at final concentrations of 250 or 25 ng/ml.

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