Immunomagnetic microbeads

Peripheral γδT cells were purified by negative selection using immunomagnetic microbeads (Miltenyi Biotec, 130‐092‐892). The non‐γδT cells were indirectly magnetically labeled with a cocktail of biotinylated monoclonal antibodies and Anti‐Biotin MicroBeads. The magnetically labeled non‐TCRγδ⁺ cells were retained in a MACS® Column in the magnetic field of a MACS Separator, while the unlabeled γδT cells passed through the column. The purity of the isolated γδT cells was greater than 95%.

Apheresis products were obtained from healthy donors (Charité ethics committee approval EA2/216/18) and peripheral blood mononuclear cells were isolated using Ficoll-Paque (GE Healthcare). CD4⁺ and CD8⁺ T cells were obtained by positive selection using immunomagnetic microbeads (Miltenyi Biotec), and activated with anti-CD3/CD28 beads (Life Technologies). On day three, activated T cells were transduced with the CAR-containing lentivirus. EGFRt⁺ CAR T cells were enriched by immunomagnetic selection with biotin-conjugated cetuximab (Bristol-Myers Squibb) and streptavidin microbeads (Miltenyi Biotec). Untransduced T cells were used as negative controls alongside CAR T cells in all experiments. CAR T cells and control T cells were cryopreserved until further use. Cryopreserved cells were thawed, stimulated with irradiated peripheral blood mononuclear cells, irradiated lymphoblastoid TMLCL cells, and OKT3 (30 ng/mL, Miltenyi Biotec), and expanded according to a rapid expansion protocol (26 (link)). CD4⁺ T cells were supplied with IL2 (50 U/μl) and IL7 (10 ng/μl) and CD8⁺ T cells were supplied with IL2 (50 U/μl) and IL15 (10 ng/μl) every other day following expansion. Functional in vitro assays were conducted between days 11 and 16 of culture.

Endothelial cells were isolated from the lungs of 4- to 6-week-old mice; although they were younger than mice used in our other experiments, we found that juvenile lungs produced higher-quality cell preparations for functional studies. As previously described, CD146⁺ pulmonary endothelial cells (PECs) were isolated with immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in MV2 medium (Promocell), supplemented with 10% fetal calf serum, 100 units/mL penicillin, and 100 mg/mL streptomycin (9 (link)). Cells were studied at passage 2 to 3.

CSCs were isolated by magnet-activated cell sorting (MACS) from the hearts of male Sprague-Dawley rats as described previously (13 (link),21 (link)). Briefly, the heart was excised and the aorta was rapidly cannulated, followed by perfusion with Ca²⁺-free Tyrode solution for 10 min and then digestion with 0.5 mg/ml collagenase (Sigma, St. Louis, MO, USA) and 0.05 mg/ml trypsin (Difco, Kansas, MO, USA) at 37°C for 30 min. The heart tissue was sectioned and the resulting cell suspension was filtered with a strainer (Becton-Dickson, Franklin Lakes, NJ, USA). Cells were then incubated with a rabbit anti-c-Kit antibody (1:50; Santa Cruz Biotechnology, Inc., Texas, USA) and separated using immunomagnetic microbeads (Miltenyi Biotech, Bergish Gladbach, Germany). CSCs were then cultured in Dulbecco’s modified Eagle’s medium/Ham’s Nutrient Mixture F12 (1:1) (DMEM/F12) (Sigma-Aldrich) containing 15% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) (both from Sigma-Aldrich) and 2.5 μ/ml erythropoietin (EPO) (BioLegend, San Diego, CA, USA) at 37°C. After 28 days of culture, confluent CSCs were passaged.