Ova solution

The ovalbumin (OVA)-Alhydrogel® dry powder vaccine was prepared as described previously (5 (link), 6 (link)). Briefly, OVA-Alhydrogel® liquid vaccine was prepared by adding 10 mL of Alhydrogel® (~10 mg/mL aluminum, manufactured by Brenntag, and supplied by InvivoGen, San Diego, CA) into a 50-mL tube followed by the addition of 10 mL of an OVA solution (1 mg/mL in 0.9% saline solution, w/v), and 200 mg of trehalose (Sigma-Aldrich, St. Louis, MO) to obtain a final formulation with 2% (w/v) of trehalose, ~1 % (w/v) of Alhydrogel®, and 0.5 mg/mL of OVA. The vaccine suspension was converted into a dry powder using our previously reported thin-film freeze-drying method (5 (link), 6 (link)). The powder was dried using a VirTis Advantage Bench Top Lyophilizer (The VirTis Company, Inc. Gardiner, NY). Lyophilization was performed over 72 h at pressures less than 200 mTorr, while the shelf temperature was gradually ramped up from −40 °C to 26 °C. After lyophilization, the solid dry powder vaccine was transferred into a sealed container and stored in a desiccator at room temperature.

Single-cell suspension of spleen and lymph nodes were obtained from OTII Foxp3-GFP mice and enriched in CD4⁺ T cells by positive selection on a MACS column (Miltenyi). Cells were then labelled with fluorescent dye conjugated monoclonal anti-TCRβ and anti-CD4 antibodies and sorted by flow cytometry using FACSAria III (BD Bioscience) as TCRβ⁺ CD4⁺ GFP⁻ cells up to >95% purity. Purified naïve T cells from OTII Foxp3-GFP mice were adoptively transferred into Zfp36^flox/flox or Zfp36^ΔDC mice. Each mouse was injected i.v. with at least 1,5×10⁶ naïve T cells in PBS. Mice were then fed a 1.5% OVA solution (Sigma-Aldrich) in drinking water for 6 days. On day 6, cell suspensions from spleen, MLN and SI LP were prepared for flow cytometry analysis.