The relative involvement of alanine-substituted HVEM(23–39) peptide analogs was assessed by ELISA (at least two experiments in triplicate) in the presence of graded peptide concentrations (5, 1 and 0.1 mg/mL). The percentage of inhibition of the BTLA/HVEM binding was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of modified peptide to the same concentration of wild-type peptide HVEM(23–39). **: p< 0.01 following non-parametric One-way ANOVA and Dunn’s post-test.