Annexin 5 assay

Freshly isolated CD8⁺ T cells (10⁶/mL) were activated with CD3/CD28 T-cell activator (25μl/ml, Stemcell, Vancouver, BC, CA) and IL-2 (150IU/ml, PeproTech, Bionity, Rocky Hill, CT, USA) in freshly-prepared RPMI for 48h. In some experiments, CD8⁺ Jurkat cells (10⁶/ml) were plated in a 96-well plate (10⁵cells/well) in MV-depleted RPMI for 24h. Next, exosomes (2–3μg in 50μL PBS) isolated from plasma of HNC patients with AD, REC or NED and from plasma of NDs were added and co-cultures were incubated for 24h. Co-cultures containing no exosomes (=50μL PBS) served as controls. Apoptosis of CD8⁺ T cells was measured by flow cytometry using an Annexin V assay (Beckman Coulter, Atlanta, GA, USA) and Accuri flow cytometer (BD Bioscience, San Jose, CA, USA). Additionally, Caspase 3/7 activity was measured in some experiments using Caspase 3/7 Glo and Cell Titer Glo Viability assays following the manufacturer’s instructions (Promega, Madison, Wi, USA) and assessed in GloMax 96 Microplate Luminometer (Promega).

Chol-DNA and DNA’-Biotin were preannealed as described above. 20 μg of exosomes were gently vortexed with preannealed solution for tethering (20 μM dsDNA tether concentration). Samples were washed with 100k MWCO filters, followed by reverse spin to get Exo-dsDNA-biotin. Exosome with 10μM biotinylated DNA as described above. 20 μg exosomes were incubated with 100ng SA-FasL for 30 min at 37°C. Post incubation, Exo-dsDNA-SA-FasL were isolated by the miniSEC method described in the exosome isolation protocol. Jurkat cells (20⁶/mL) were cultured in freshly prepared RPMI-1640 medium supplemented with 10 % ED-HI-FBS for 48 hrs. 20 μg exosomes decorated with 100ng (dsDNA)SA-FasL were added to the media and incubated for 12 hrs. Native exosomes (non-engineered exosomes) and Exo-dsDNA-biotin were used as controls. Apoptosis of Jurkat cells was measured by flow cytometry using an Annexin V assay (Beckman Coulter, Brea, CA).