Figure 3.

The 7D12-5GS-6H4 bispecific VHH induces Vγ9Vδ2-T cell activation and lysis of EGFR expressing tumor cells. Vγ9Vδ2-T cells were cultured with or without EGFR⁺ A431 tumor cells (A-B) or EGFR⁻ JY cells (B) in a 1:1 ratio in the presence of the 7D12-5GS-6H4 bispecific VHH or a bispecific control VHH. VHH concentrations: (A) 10 nM; (B) as indicated. For control situations, Vγ9Vδ2-T cells were co-cultured with target cells in the absence of VHH (no VHH; negative control) or with NBP-pretreated target cells (positive control). After 24 hrs, Vγ9Vδ2-T cell activation and degranulation was determined by assessing the percentage of CD25 or CD107a expression, respectively by flow cytometry. The percentage of lysed target cells was determined using 7-AAD staining and flow cytometry. A) White bars represent Vγ9Vδ2-T cell mono-cultures in the absence of VHH, grey bars represent target cell mono-cultures in the absence of VHH and black bars represent Vγ9Vδ2-T cell co-cultures with target cells and indicated VHH. B) Co-cultures of target cells with Vγ9Vδ2-T cells and indicated amount of 7D12-5GS-6H4 bispecific VHH. Shown are mean ± SEM of n = 3-4 experiments. p-Values were calculated with a one-way ANOVA and Bonferroni's post-hoc test (* indicates p<0.05 and *** indicates p<0.001). Abbreviations: aminobisphosphonates (NBP); Gly₄Ser (GS).