Ficoll paque gradient separation

50-days-old Tg2576 mice and WT littermates were i.v. injected with 150 µL of WT or Tg2576 blood from 12 to 14 months old mice. Groups of animals (n = 4) were sacrificed 1.5 h or 7 days after the treatment whereas others were sacrificed 1.5 h or 7 days after a second blood transfusion occurring 30 days later. Plasma and peripheral blood mononuclear cells (PBMC) were obtained from blood of treated and non-treated Tg2576 and WT mice at the indicated times post-transfusion. Plasma and PBMC were isolated by Ficoll-paque gradient separation (GE Lifesciences, Little Chalfont, UK). 1 × 10⁶ PBMC/mL were incubated for 15 h with 100 ng/mL of lipopolysaccharides from Escherichia coli (LPS, Sigma-Aldrich, St. Louis, MO) diluted in RPMI Medium 1640 (Sigma-Aldrich, St. Louis, MO) and supplemented with 10% fetal bovine serum. Supernatants were recovered and stored at − 80 °C until use. IL-1β, IL-6, TNF-α, MIP-1α, IFN-γ, IL-10 and MCP-1 in plasma and PBMC supernatants were measured by the respective Quantikine Kit (R&D Systems, Minneapolis, MN), according to manufacturer’s specifications. Signals were read on an EL800 BIO-TEK ELISA plate reader (BioTek, Winooski, VT) at 450 nm. The same experimental procedures were performed in animals injected with different blood components (n = 4–6). Samples in this case were obtained at the experiment’s endpoint (~ 200 days after the first inoculation).

White blood cells from healthy donors were obtained from the Blood Center of Shanghai (Shanghai, China). Human PBMCs were isolated from whole blood by Ficoll-Paque gradient separation (GE Healthcare, Boston, MA, United States). CD3⁺ T cells were enriched by negative immunomagnetic bead selection according to the manufacturer’s protocol (Miltenyi Biotec, Germany). Isolated CD3⁺ T cells were stimulated with CD3/CD28 T-cell activation Dynabeads (Gibco) for 48 h. For infection, 1 × 10⁶ stimulated CD3⁺ cells were transduced with concentrated pseudovirus supernatant (MOI = 10) plus 7 μg/ml polybrene (Yeason) for 12 h, and then, the second round of infection was carried out with the same procedure. T cells were expanded in complete T cell medium containing 90% vivo15 (Lonza, Basel, Switzerland) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. Cells were also fed with 5 ng/ml IL-2 (R&D, St. Paul, MN, United States), 2 ng/ml IL-7 (R&D) and 2 ng/ml IL-15 (R&D) every 48 h. 4-7 days after LV transduction, CAR⁺ cells were enriched by staining with FITC-conjugated goat anti-human F(ab’)₂ antibody (Jackson ImmunoResearch Laboratories, West Grove, United States) and sorted by a FACS Fusion I (BD Biosciences, Grand Island, NY, United States). Genetically modified T cells were used for functional assays after sorting.