Mifn γ

For the staining of surface proteins, cells were resuspended in FACS buffer (0.1% BSA in PBS) with the respective antibodies and incubated for 30 minutes on ice and in the dark. The following antibodies were used: mCD4 (eBioscience), mCD8 (BD Biosciences), mCD45.1 (eBioscience), mCD45.2, mIFN-γ, mTNF-α (all from BD Biosciences); hCD4 (Thermo Fisher Scientific), hCD8 (Dako). Viability of cells was determined using a live/dead cell marker (LIVE/DEAD Fixable Green Dead Cell Stain Kit, Thermo Fisher Scientific). For intracellular cytokine staining, cells were permeabilized by resuspending them in Cytofix/Cytoperm (BD Biosciences) prior to the antibody staining, following the manufacturer’s instructions. In order to determine the absolute cell count by flow cytometry, CountBright™ Absolute Counting Beads (Thermo Fisher Scientific) were added to the cell suspension shortly before measurement. If CAR expression was analyzed together with other surface proteins such as CD8, stainings were performed sequentially. The CAR was first stained with an anti-human IgG antibody (Abcam) followed by the staining of the other surface proteins with the respective antibody. The samples were then analyzed on a CytoFLEX S flow cytometer (Beckman Coulter). The obtained data was evaluated with FlowJo 10.4.