*4.5. Blood, Spleen and Lymph Nodes Collection and Processing*

At day 24, animals were maintained under general anesthesia with volatile isoflurane and whole blood was collected by cardiac puncture into tubes containing anticoagulant citrate-phosphate-dextrose solution (Sigma-Aldrich Inc., St Louis, MO, USA). Blood was centrifuged, and plasma and buffy coats collected and processed as described previously [41]. Plasma was further centrifuged twice at 2500× *g* for 15 min and supernatant stored at −80 ◦C until further analysis. Collected buffy coats were diluted in phosphate buffered saline (PBS), overlaid on Lymphoprep (Axis-Shield Diagnostics, Dundee, Scotland) in a 1:1 ratio and centrifuged at 800× *g* for 30 min at RT to isolate peripheral blood mononuclear cells (PBMC).

Animals were dissected for the collection of spleen and draining lymph nodes (inguinal and popliteal nodes). Spleen was clean from adjacent tissue and weighted, and then a half portion of spleen and lymph nodes were preserved in formalin 10% for further histology processing, whereas the remaining parts were used to isolate single cells for flow cytometry analysis, as described previously [41]. Briefly, spleen was digested with 1 mg/mL Collagenase I (Sigma-Aldrich Inc., St Louis, MO, USA), both spleen and lymph nodes were gently crushed, and strained to obtain cell suspensions. Red blood cells in spleen cell suspension were then lysed by incubation with NH4Cl 150 mM in Tris 10 mM. Obtained cells were washed with PBS.
