Since the cyclic peptide C29-C37, where cysteine residues were oxidized, does not block the BTLA/HVEM binding, we wondered whether the blocking ability of HVEM(23–39) fragment may be due to the capacity of HVEM-based peptide cysteines to reduce the cysteine disulfide bridges present on the BTLA surface, or possibly make disulfide bridges with these BTLA cysteines instead. Therefore, the effect of free cysteine and methionine amino acids or oxidized dimer of cysteine (cystine) on the BTLA-HVEM binding was analyzed. Results showed that cysteine with free sulfhydryl group targeted BTLA protein and prevented partially the binding of HVEM, whereas for the methionine and cystine, the inhibitory properties were not observed (Fig 6A). An Ellman’s assay was then performed on both HVEM(23–39) peptide and recombinant BTLA-Fc in order to quantify free thiol groups. In contrast to BTLA-Fc, we observed free thiol groups in the HVEM(23–39) peptide. However, after 2 hours of incubation, the concentration of free thiol groups in HVEM(23–39) peptide was lower, suggesting that a fraction of the peptide is oxidized. Besides, when the HVEM(23–39) peptide was incubated with BTLA-Fc, we also observed a decrease in the concentration of free thiol groups over time, which was more pronounced than in the condition where the peptide was alone (Fig 6B). These results suggest that BTLA protein may form disulfide bonds with the HVEM(23–39) peptide, lowering the amount of free thiol groups in the peptide.