Ack solution

Popliteal and inguinal LNs were isolated and a single-cell suspension was prepared by incubating LN with 1 mg/mL collagenase D (Roche) and 0.04 mg/mL DNase I (Boehringer) in 5% FSC containing DMEM, for 30 min at 37°C. Organ pieces were passed through a 70-μm cell strainer and stained for cell-specific markers. The following fluorochrome-labeled antibodies were used: CD11c, CD8a (all from eBioscience), F4/80, Live/Dead Aqua cell stain (all from Life Technologies).
Spleens were isolated smashed through a 70-μm cell strainer using the plunger of a syringe (Falcon), red blood cells were lysed with ACK solution (Lonza). The following fluorochrome-labeled antibodies were used: CD3, CD8a, Live-Dead dye, IFNγ, TNFα, and IL-17 (eBioscience).
Primary culture of DCs from LNs of a naïve mice were harvested and incubated for 24 h with 10 nM of E7_49-51 conjugated with Alexa488 and 1 μg/mL of Qβ conjugated with Alexa647. CD11c⁺ F4/80⁻ DCs were subsequently analyzed by flow cytometry for uptake of peptide and Qβ.

T-cell absolute count was performed on blood samples kept from the mandibular vein of the mouse. To evaluate T lymphocyte number, we used an anti-mouse CD3 PE conjugated antibody (BD Biosciences, San Jose, CA, USA, clone 145–2C11). At predetermined optimal concentrations, 100 μL of blood was stained by incubation with the antibody. Fifty microliters of Count Bright Absolute Counting Beads (Molecular Probes, Milan, Italy) were added, and erythrocytes were lysed using ACK solution (Lonza Bio Whittaker, Walkersville, MD, USA), according to standard protocols. Cell suspensions were acquired and analyzed on LSR Fortessa™ X20 SO Cell Analyzer (BD Biosciences). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated. In particular, the formula used was: (number of cells counted/number of beads counted) × (lot-specific number of beads/sample volume).