Pma ionomycin mixture

Single-cell suspensions of tumor-infiltrated immune cells were prepared. Cells were incubated with anti-CD16/CD32 antibody (BD Pharmingen, #553141) to minimize non-specific binding. All cell surface reactions (CD45, CD3, CD4, CD8, CD25, CD206, I-A/I-E, CD11c, CD11b, F4/80, PD-1, CD86, Ly-6G, and Ly-6C) were performed at 4 ℃ for 30 min. Activated human primary T cells (Stemcell Technologies) were treated for 24 h and 4 h, respectively, with a PMA/ionomycin mixture (MultiSciences, Hangzhou, China, #70-CS1001) and brefeldin A (BFA) (Sigma-Aldrich, MO, USA, #B5936) as pretreatment. The T cells were fixed and permeabilized using the Fixation/Permeabilization Set protocol (eBioscience, CA, USA, #00-5123-43, #00-5223-56, #00-8333-56) and incubated with IFN-γ antibody (BD Pharmingen, #554700) in the dark for 30 min. The permeabilization step allowing intracellular staining (KI-67, IFN-γ, GZMB, and Foxp3) was performed referring to the manufacturer’s protocol (BD Pharmingen, #562574, #550583). The gating strategy is shown in Additional file 3: Fig. S3. All the tumors in each group were detected. In addition, when using flow cytometry to measure the abundance of PD-L1 expression on the surface of pancreatic cancer cells, each group examined three independent samples.

The fluorescent mAbs CD4, CD19, CD11c, F4/80, CD1d, CD5 and IL-10 were purchased from Elabscience (Wuhan, China). Intracellular staining was conducted as described previously (Yang et al., 2012 (link)). Splenocytes from mice were suspended in the presence of PMA/ionomycin mixture (Phorbol 12-myristate 13-acetate, Multisciences) and monensin (Multisciences) for 5 hours to analyze B10 cells (CD1d^hiCD5⁺B19⁺IL-10⁺). Then, the cells were collected and stained with PE Anti-CD19 mAbs, FITC anti-CD1d mAbs and PerCP/Cyanine5.5 anti-CD5 mAbs. After removing the unbound antibodies, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences, San Jose, CA). Then, they were stained with APC mouse anti-IL-10 following the manufacturer’s instructions. The samples were performed using BD FACSCanto flow cytometer (BD Biosciences), and the results were analyzed using FlowJo Software (Version X; TreeStar, Ashland, OR).