293T cells expressing BTLA (generous gift from Dr. C. Krummenacher, University of Pennsylvania, USA) were maintained in culture with RPMI 10% FCS supplemented with 0.2 mg/ml hygromycin B. 2x105 293T cells expressing BTLA were incubated with rhHVEM-Fc at 8μg/ml in PBS 0.5% BSA for 30min at 4°C. Cells were then washed and incubated with goat anti-human Fc conjugated with AF647 for 20min at 4°C (Jackson ImmunoResearch, #109-606-170). Inhibition of HVEM-BTLA binding by different peptides was assessed by pre-incubating the cells with the different peptides at 5 mg/ml for 1h at 4°C. BTLA expression was controlled by anti-BTLA APC-conjugated antibody (Biolegend, #344510). Maximum inhibition of binding was determined by adding anti-BTLA7.1 antibody for 1h at 4°C at 2μg/ml before adding rhHVEM-Fc. Sample acquisition was performed on the Gallios Flow-Cytometer (Beckman Coulter) and data were analyzed using the FlowJo Software (TreeStar).