Recently, activatory and inhibitory immune receptors and their ligands emerged as promising targets for cancer immunotherapies. Indeed, treatment based on the blockade of immune checkpoints such as PD-1 or CTLA-4, have demonstrated major clinical benefit for cancer patients [40–42]. It is known that BTLA is involved in the negative regulation of T-cell responses by interacting with HVEM [27, 43], highlighting BTLA as a potential target for cancer immunotherapy. Our study focused on the design of blocking peptides of the interaction between BTLA and HVEM, based on the amino acid sequence of HVEM binding fragment. The crystal structure of BTLA/HVEM complex indicated that in the interaction between the two proteins, two fragments of BTLA: (35–43) and (118–128) bind with HVEM: (26–33) and (33–38) respectively. Therefore, HVEM(23–39) fragment was synthesized and its interaction between BTLA and HVEM(23–39) was confirmed by using affinity chromatography. The competitive ELISA tests showed also that the peptide could inhibit the proteins binding. However, we showed using competitive ELISA and cellular assay that this blocking capacity was mainly due to cysteine residues present in the peptide sequence or in solution. Compaan and co-workers showed that the interactions between BTLA and HVEM proteins are primarily stabilized by main chain hydrogen bonds and includes relatively few side chain contacts . The conformation of the HVEM(23–39) peptide is significantly different than the conformation of the same fragment in the native protein due to the fact that it is not stabilized by disulfide bridges and the peptide poses two cysteine residues with free sulfhydryl groups. In the monomeric crystal structure of the extracellular domain of BTLA protein, six cysteine residues form three disulfide bonds between residues 72–79, 34–63, and 58–115. The Cys58–Cys115 disulfide bond is completely buried (solvent accessible surface is 0% for both cysteine) in the hydrophobic core of BTLA . The Cys58–Cys115 (solvent accessible surface is 2,9% and 17,9%) and more Cys34–Cys63 (solvent accessible surface is 45,1% and 33,3%) disulfide bonds are exposed to the solvent and could be sensitive for the other, free or high reactive sulfhydryl group. Therefore, we propose that free sulfhydryl groups of HVEM(23–39) peptide or free cysteine amino acid compete, in our in vitro assays, with disulfide bridges (Cys58–Cys115 or Cys34–Cys63) of the BTLA protein to form covalent S-S bond between HVEM(23–39) peptide or cysteine amino acid and BTLA protein. This may most likely change the structure of BTLA, thus hindering the binding of HVEM. Yet, we observed that the scrambled peptide, which also contains free thiol groups as confirmed by Ellman’s assay (S2 Fig), lacks blocking activity. In order to reduce the disulfide bonds on BTLA, peptides have to pre-dock to the surface of the protein. We hypothesize that scrambled peptide may have a lower affinity to BTLA, compared to HVEM(23–39) peptide. As a consequence of the weaker binding to BTLA the scrambled peptide may not be able to reduce the exposed disulfide bonds of BTLA and form disulfide bonds with the protein.