The analogs (Table 2) were tested in the ELISA tests and compared to the HVEM(23–39) and scrambled peptide (Fig 5). We found that both substitutions of Cys29 significantly abrogated the blocking capacity of the HVEM(23–39) peptide. This could indicate the lack of specific interactions of Cys29 residue and point to the importance of the free sulfhydryl group for the binding of peptides. Moreover, albeit not significant, the shorter synthesized peptide: HVEM(31–39) that lacks the Cys29, showed a lower percentage of inhibition than the HVEM(23–39). To further test the importance of Cys37, we synthesized and tested the its analog (HVEM(31–39) C37S). We found that this peptide had no blocking capacity. Finally, the cyclic peptide HVEM(23–39) (C29-C37), encompassing blocked sulfhydryl groups, was also tested, but no blocking of BTLA/HVEM interaction was observed (Fig 5). Of note, in CD spectrum, formation of the disulfide bridge in HVEM(23–39) peptide did not significantly change the peptide structure (S1 Fig).