Tigit fc

Streptavidin Dynabeads (Thermo Fisher Scientific) were coated with either 200 pmol biotinylated protein or 10 μg biotinylated IgG/mg Dynabeads. Ligand-screening was performed by coating with biotinylated CD155 (PVR) (AcroBiosystems), CD112 (Nectin-2) (BPSBiosciences) or biotin as a negative control and anti-KIR2DL5 as a positive control. To screen for interactions with HLA class I and class II molecules, the LABScreen Single Antigen Class I and II kits (OneLambda) were used. Negative control beads were not coated with HLA antigens and positive control beads were coated with purified human IgG. Recombinant human Fc constructs (KIR2DL1-Fc, KIR2DL3-Fc, KIR2DL4-Fc, KIR2DL5-Fc, KIR3DL1-Fc, CD96-Fc, DNAM-1-Fc, LAG-3-Fc, PVR(CD155)-Fc, TIGIT-Fc) (R&D Systems) were diluted to 250 μg/ml in PBS and co-incubated with coated beads for 15 min at 4°C at a final concentration of 25 μg/ml. Samples were washed and bead-bound Fc constructs were detected by a staining with F(ab)2 goat-anti-human IgG PE secondary antibody (Life Technologies) for 15 min at 4°C. Interactions between Fc construct and peptide-coated beads were either quantified by flow cytometry (LSR Fortessa (BD Biosciences)) or by using the Luminex xMAP technology on a Bio-Plex 200 (Bio-Rad Laboratories).

Mice were immunized with bacterially generated recombinant Tax protein and CpG by subcutaneous inoculation at days 0 and 14, and splenocytes were collected at day 21. Splenocytes of immunized mice and human PBMCs were subjected to ELISPOT assay as described [56 (link)]. Recombinant mouse TIGIT-Fc and mouse lgG2A-Fc (R&D) were coupled to Dynabeads M-450 (Invitrogen) according to the manufacturer’s protocol. Cells were stimulated with Tax peptides along with the Fc-conjugated beads or blocking antibodies as indicated.