E2620

A volume of 100 µl of luciferase expressing cells were transferred into 96-well plates. Cells were mixed with an equal volume of bright-glo assay solution (Promega, E2620) and incubated at room temperature for 10 min with shaking. Half of the mixture was transferred into a 96-well white opaque plate. The luminescence signal was read using an EnSpire alpha (PerkinElmer) detector.

For Jurkat cell stimulation assay shown in Fig 2E and F, Appendix Fig S1C and Fig 5C, Jurkat‐PD‐1 cell transfected with NFAT‐luciferase reporter (designated JP‐luc) was generated. Raji stably expressing hPD‐L1 was generated (designated Raji‐L1) as antigen‐presenting cells (APC). Raji cells or Raji‐L1 cells were preincubated with 1 μg/ml of SEE superantigen (Toxin Technology, ET404) in serum‐free RPMI medium for 1 h at 37°C. JP‐luc cells were pretreated with DMSO, MB, or aPD1 antibody for 1 h before mixing with SEE‐loaded Raji or Raji‐L1 cells at ratio of 1:1 in a 96‐well plate for 6 h. Luciferase activity was measured with a luminometer following the manufacturer's instructions (Promega, E2620).
For Fig 2H, JP‐luc cells were co‐cultured with Raji‐L1 or parental Raji cells in a 96‐well plate precoated with 10 μg/ml of aCD3/aCD28 for 6 h and the supernatants were collected after 24 h. IL‐2 was quantified by ELISA using Human IL‐2 ELISA kit (Mlbio, HX2390).