Tetanus Toxoid Bystander Suppression Assay (TTBSA)

The TTBSA measures the inhibitory capacity of potential regulatory peptides on the recall response of human CD4 T cells to the tetanus toxoid (TT) antigen was adapted for validation of Treg epitopes and previously described (27, 31). TT vaccination is a routine, nearly universal immunization, resulting in memory T cell responses that persist for many years (32). Therefore PBMCs are considered to be a reliable source for in vitro assays that require TT-specific memory T cells.

Briefly, PBMCs were labeled with CFSE cell proliferation dye (eBioscience) and rested overnight at 37°C, 5% CO₂. The following day cells were stimulated with Tetanus toxoid (TT) (Astarte Biologics, cat no. 1002) at 0.5 μg/ml alone and in combination with the putative regulatory peptides or control peptide at 8, 16 or 24 μg/ml, then incubated for 6 days and analyzed by flow cytometry on day 7. CD4+ T cell proliferation, T effector activation and the ratio of regulatory to effector T cells were measured.