293T cells expressing human BTLA were incubated with peptides or free amino acids (5mg/mL) prior labeling with rhHVEM-Fc and AF647-conjugated anti-human IgG antibody (at least two experiments in triplicate). The graph shows the Geometric Fluorescence Intensity (GMFI). *: p< 0.05 and ***: p< 0.001 following non-parametric One-way ANOVA and Dunn’s post-test.