Cytofix cytoperm method

Cells were stained for EGFRvIII⁺ CARs to detect cell-surface expression using an EGFRvIII multimer-PE as previously described [50] . Negative staining controls were conducted by staining untransduced cells from the same donor. ICS was performed by co-culturing T cells with tumors 1∶1 over 18 hours with the BD GolgiPlug protein transport inhibitor containing brefeldin A (BD Sciences, San Jose, CA) in RPMI-1640 medium plus 10% FBS. Following co-culture, cells were submitted to surface staining for CD3, CD8 and intracellular IFN-γ stain using the BD Cytofix/Cytoperm method (BD Biosciences, San Jose, CA).

Human albumin (cat# E80–129 Bethyl Laboratories), human IgM (cat# 109–035–043 Jackson ImmunoResearch), and human IgG (cat# 109–035–008 Jackson ImmunoResearch) levels were quantified in the plasma by ELISA according to the manufacturer’s instructions and previously described protocols [16 (link)]. Mononuclear cells from blood, spleen, bone marrow, thymus and lymph nodes were isolated as previously described [16 (link)]. Liver leukocytes were isolated after eliminating circulating cells by perfusing the liver with PBS in situ through the vena cava as previously described [28 (link)]. To test the functionality of T cells, splenocytes were cultured in IMDM with 10% fetal calf serum, 10 IU/ml hIL-2 (Chiron), 100 ng/ml hIL-7 (Miltenyi), 4x10⁵ Dynabeads CD3/CD28 (Life technologies) for 9 days then stimulated for 4 hours with 50 ng/ml PMA and 1 microg/ml ionomycin for 4 hours prior to intracellular cytokine staining using the BD cytofix/ cytoperm method (BD biosciences). Flow cytometry was performed with directly conjugated antibodies according to standard techniques using a Fortessa and LSRII flow cytometers (Becton Dickinson). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) and a 405 nm excitation were used to exclude dead cells. The list of anti-human antibodies used in flow cytometry is detailed in S1 Table. Analysis was performed with Flowjo Version 8.8 (TreeStar).