In order to be in a more physiological setting, we developed a cellular assay, where 293T cells expressing BTLA were stained with rhHVEM-Fc followed by AF647-conjugated anti-human IgG antibody. Using this assay, we tested HVEM(23–39) fragment as well as HVEM(23–39) (C29-C37), scrambled peptide (Ctrl), cysteine and methionine amino acids. Surprisingly, in contrast to ELISA tests, almost no blocking of the BTLA/HVEM binding was observed with HVEM(23–39) peptides. However, the free cysteine amino acid blocked BTLA/HVEM complex formation (Fig 7).