Rat serum

For flow cytometry, 1 × 10⁶ cells were used per sample unless otherwise indicated. A mix of 0.05 mg/mL purified rat anti-mouse CD16/CD32 (BD Pharmingen), rat serum (GeneTex, Irvine, CA) and hamster serum (Jackson Immunoresearch, West Grove, PA) was used to block Fc receptors on tumor infiltrating leukocytes for 20 min at 4 °C. This step was omitted for tumor cell lines and splenocytes. The cells were then washed twice and 100 μL of LIVE/DEAD® Fixable Yellow Dead Cell Stain or LIVE/DEAD® Fixable Blue Dead Cell Stain Kit, for UV excitation (Life technologies / Thermo Fisher) diluted 1:1000 in PBS was added. After incubation for 30 min at 4 °C, washed cells were stained with antibodies (or isotype controls) listed in Additional file 1: Table S1. After incubation for 1 h at 4 °C, cells were washed and resuspended in 200–400 μL FACS buffer for flow cytometric sorting or analysis, respectively. For intracellular cytokine staining, cells were incubated in 100 μL fixation permeabilization solution (BD Biosciences, San Jose, CA) for 20 min at 4 °C, followed by two washing steps using BD Perm/Wash buffer. Cells were then incubated for 1 h at 4 °C with 100 μL BD Perm/Wash buffer containing antibodies or isotype controls, respectively, diluted 1:100. Finally, cells were resuspended in 200–400 μL FACS buffer for analysis.

Immunofluorescence staining was performed using monoclonal antibodies shown in Table S2. As controls, the respective isotype matched antibodies against irrelevant epitopes were included. Cells (2 × 10⁵) were incubated at 4°C for 20 min in a mixture of rat anti-mouse CD16/CD32 (Becton Dickinson), normal Syrian hamster serum (Jackson Laboratory, Bar Harbor, USA) and rat serum (GeneTex, Irvine, USA) in a total volume of 100 μl to block Fc-receptors. Subsequently, cells were incubated with fluorochrome conjugated antibodies diluted in PBS containing 3 % FCS and LIVE/DEAD Fixable Yellow Dead Cell dye (1:1000) at 4°C for 1 h. In case of intracellular staining cells were fixed and permeabilized using FoxP3 staining Kit (eBioscience, Waltham, USA) according to manufacturer's instructions. Thereafter, the cells were incubated with fluorochrome conjugated antibodies diluted in Permeabilisation Buffer (Thermo Fisher Scientific) at 4°C for 1 h. Finally, cells were analyzed with a FACSCanto II or LSR II (Becton Dickinson) cytometer or sorted using a FACS Aria I or II and data were evaluated with FlowJo software (Version 10).