Pma ionomycin activation cocktail

Organs were mechanically disrupted using a sterile syringe plunger through a 70 μm nylon strainer (Falcon, Catalog #352350) to obtain single cell suspensions. Single cell suspensions were resuspended in 50 μL of surface antibody staining solution, diluted in PBS + 2% FBS + 1 mM EDTA (PBSFE), and incubated for 60 min at 4 °C on a shaking platform. Cells were washed, fixed and permeabilized. Finally, cells were stained with the intracellular target antibodies overnight (12–16 h) in antibody solution diluted in permeabilization buffer (Biolegend, Catalog #424401). For intracellular cytokine detection, single cell suspensions were resuspended in 100 μL of RPMI1640 + 5% FBS and PMA/ionomycin activation cocktail (Biolegend, Catalog # 423302) and incubated at 37 °C, 5% CO₂. After 1 h, 10 μL of a 10× solution of Brefeldin A (Biolegend, Catalog #420601) and Monensin (Biolegend, Catalog #420701) diluted in RPMI1640 + 5% FBS were added and cells were incubated for an additional 4 h. Next, cells were stained intracellularly as described above. Samples were acquired on a BD Fortessa flow cytometer. Antibodies were purchased from Biolegend and BD Biosciences and were used at optimized dilutions. Illustrative flow cytometry gating is show in Supplementary Fig. 16. Antibody-fluorophore combinations, clones and dilution are reported in Table S2.

CD4⁺ OT-II and CD8⁺ OT-I cells were isolated as previously described. A mixture of 1 × 10⁶ CD4⁺ OT-II and 4 × 10⁵ CD8⁺ OT-I cells was injected i.v. into recipient wild-type mice. One day later, mice were immunized s.c. with 50 μl of the indicated NP into both flanks. Spleens were harvested 5 days p.i. Tissues were processed to yield a single cell suspension. After lysis of red blood cells, cell suspensions were washed and filtered through a 70 μm cell strainer. Cells were then incubated O.N. at 37°C, 5% CO₂ in RPMI (Biological Industries) and stimulated with PMA/Ionomycin activation cocktail (1:2000, BioLegend), Brefeldin A (0.5 μg/ml, BioLegend) and OVA peptide (323-339, 20 μg/ml, GenScript). On the following day cells were stained for extracellular TCRβ, CD4, CD45.1, CD44 and intracellular IFNγ and IL-17A according to the guidelines of the Cytofix/perm kit (BD). Cells were analyzed by flow cytometry.