Construction of 2B4 NFAT–GFP reporter cells

2B4 NFAT–GFP cell line is a T cell hybridoma cell line in which GFP expression is controlled by the transcriptional factor NFAT. LAIR‐1–CD3ζ construction has been described [15], and SIRL‐1–CD3ζ construction was performed similarly, yielding a 269 AA chimera comprising the SIRL‐1 signal peptide and ectodomain (MTAEFLSLLC…APSMKTDQFK) fused to the CD3ζ transmembrane and cytoplasmic domains (LCYLLDGILF…ALHMQALPPR). The construct was transduced into a 2B4 NFAT–GFP cell line, in which ligation of the chimera by an antibody or a ligand results in NFAT promoter‐driven GFP expression [24]. Three days after transduction, cells were sorted for high SIRL‐1 surface expression and subcloned by limiting dilution. We selected one clone with high surface SIRL‐1 expression and high GFP expression levels after SIRL‐1 ligation (“2B4 NFAT–GFP reporter cell assay” below) for further experiments. All cells were maintained in RPMI 1640 with 10% heat‐inactivated fetal bovine serum (FBS) and 50 U/ml penicillin–streptomycin (culture medium) unless stated otherwise.