Suppression Assay

As responder cells, human naïve T cells were sorted from allogenic donors as described above. Responder cells were rested overnight in resting medium (RPMI Medium 1640 + GlutaMAX supplemented with 10% FBS and 100 U/ml penicillin/100 μg/ml streptomycin).

Responder cells were stained with CellTrace Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to manufacturer’s recommendations. Fifty thousand responder cells were mixed with 50,000 suppressor cells (cultured Tregs or mCherry+ “TET1” or mCherry- or MOCK transfected on day 9 post transfection) in a 96-well round bottom plate in resting medium. One hundred thousand Treg Suppression Inspector beads (human, Miltenyi Biotec) were added. As controls, 100,000 responder cells with (“responder only act.”) and without beads (“responder only”) were cultured. On day 5 after seeding (= day 14 after transfection), proliferation of the responder cells was assessed by dilution of the CellTrace dye using flow cytometry (MACSQuant instrument, Miltenyi Biotec).