Mcherry lifeact 7 plasmid

HeLa cells were incubated at 37 °C and 5% CO₂ in a humidified atmosphere and maintained in DMEM complete medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% L-Glutamine (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were transiently transfected using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific). Briefly, cells were seeded in a six-well plate the day prior to transfection in complete medium. At 70–80% confluency, cells were co-transfected with 1 µg of the mCherry-Lifeact-7 plasmid (Addgene) together with wild-type or mutant pEGFP_YARS1 plasmid (1:1 ratio). The next day, cells were reseeded in 35 mm glass bottom imaging dishes (MatTek Corporation). Fixation (15 min 4% PFA, Laborimpex) or live cell imaging was performed the next day (48 h after transfection) on a Zeiss LSM700 laser scanning confocal microscopy. Fixed cells were imaged with Plan-Neofluar 40×/1.30 Oil objective. Time lapse imaging of living cells was done at 2fps with a Plan-Apochromat 63×/1.40 Oil objective and using the line switching mode to avoid any fluorescence channel crosstalk and to minimize acquisitional delay between channels. Images in Supplementary Fig. 6 and Supplementary Movie 2 are 332 × 268 with 85 nm pixel dimension. Channels were combined and movies were annotated with the Fiji distribution of ImageJ.

To evaluate conjugate formation by confocal microscopy, PD-1–transduced or untransduced Jurkat cells were transfected with DMRIE-C reagent and mCherry-LifeAct-7 plasmid (Addgene) according to manufacturer's recommendations. On next day, 1–2 × 10⁶ Raji B cells were coated with 2 μg/ml SEE in FCS-free RPMI for 2 h at 37 °C, 5% (v/v) CO₂. Next, 2 × 105 Raji B cells prepared in 200 μl were mixed with 100 μl of untransduced Jurkat T cells or PD-1–transduced Jurkat clone. The coculture was placed on a glass bottom culture dish (MatTek Corporation) and rested at 37 °C for 30 min to allow conjugate formation. Following, confocal images from each stimulation were acquired on Zeiss 710 confocal microscope and analyzed with ZEN software.