Fetal calf serum (fcs)

GL261 were provided by A. Fontana, Experimental Immunology, University of Zurich, Zurich, Switzerland and cultured in DMEM (Gibco) supplemented with 10% FCS (Biosera), GlutaMAX (Gibco), and sodium pyruvate 1 mM (Gibco). B16.F10 were bought from ATCC and cultured in RPMI (Gibco) supplemented with 10% FCS (Biosera) and GlutaMAX (Gibco). To express EGFRvIII or GD2, cells were transduced in six-well plates with the addition of 3 mL of a γ-retroviral vector in the presence of 10 µg/mL Polybrene (Sigma). The γ-retroviral vector was produced by transient triple transfection of HEK293T using GeneJuice transfection reagent (Merck Millipore) with 4.69 μg of Peq-Pam plasmid (encoding Moloney GagPol), 3.13 μg of VSV-G envelope, and 4.69 μg of retroviral backbone SFG expressing the gene of interest, EGFRvIII or GD2 and GD3 synthase^{55 (link)} (AddGene plasmid #75013), respectively. Supernatants containing retroviral vector were collected at 48 and 72 h post-transfection and frozen at −80 °C prior to use for GL261 or B16.F10 transduction.

Anonymized buffy coats were obtained from healthy blood donors at the Irish Blood Transfusion Service, Dublin, Ireland. Peripheral blood mononuclear cells (PBMCs) were isolated, and CD4⁺ cells were purified (CD4⁺ T cell isolation kit; Miltenyi Biotec) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as previously described (5 (link)). PBMCs were gamma irradiated at 30 Gy with a ¹³⁷Cs source (Gammacell 3000 Best Theratronics). CFSE-labeled CD4⁺ cells (1 × 10⁵) were cocultured with irradiated PBMCs (APCs) (1 × 10⁵) with cRPMI alone (negative control), staphylococcal enterotoxin A (100 ng/ml [positive control]), heat-inactivated S. aureus (1 μg/ml ≈ 1 × 10⁷ CFU/ml), or purified staphylococcal antigens (0.88 μM). cRPMI comprised RPMI (Sigma), 10% (vol/vol) fetal calf serum (Biosera), 100 mM l-glutamine (Gibco), and 100 μg/ml penicillin-streptomycin (Gibco). T cell proliferation and cytokine production were assessed on day 10 by flow cytometry.