Cell stimulation mixture

Splenocytes were stimulated with MHC class I–restricted LCMV-GP_33–41 (2 μg/mL) or MHC class II–restricted LCMV-GP_61–80 peptide (5 μg/mL) for 1 h in the absence of brefeldin A and then 5 h in the presence of brefeldin A (4 μg/mL; Sigma-Aldrich). Cells were fixed and permeabilized with 2% saponin, and intracellular staining was performed with antibodies to IFN-γ (XMG1.2), TNF-α (MP6-XT22), and IL-2 (JES6-5H4). For polyclonal stimulation, single-cell suspensions were stimulated with eBioscience Cell Stimulation Mixture according to the manufacturer’s protocol. Intracellular IL-21 staining was performed as previously described (18 (link)).

Lymphocytes were harvested from tissues and plated with or without 1× PMA and ionomycin mixture (eBioscience, Cell Stimulation Mixture), or 100 nM SIINFEKL peptide with 5 × 10⁵-labeled splenocytes for 4 to 6 h, followed by intracellular cytokine staining.

The tumor single-cell suspension was prepared as above described. For antigen-specific stimulation, the cells were plated in 24-well plates and pulsed with TRP2_180–188 (SVYDFFVWL) for 16 h at 37 °C in complete T cell media (RPMI 1640, 10% FBS, 50 μM β-mercaptoethanol, 100 U/mL Pen/Strep, 1× MEM Non Essential Amino Acids Solution, 1 mM sodium pyruvate) with ebioscience Protein Transport Inhibitor Mixture. For broad stimulation, the cells were plated in 24-well plates in complete T cell media with ebioscience Cell Stimulation Mixture (plus protein transport inhibitors) for 5 h. After stimulation, the cells were collected, washed, blocked with Fc blocker, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain and surface markers (CD45, CD3, TCRb, CD4, CD8), and then fixed using the ebioscience Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer’s instructions. Cells were then washed and permeabilized for intracellular staining for IFN-γ, TNF-α, Foxp3, and IL-17. Then, cells were analyzed on a BD LSRII flow cytometer.