Fig. 4.

Costimulatory effects of the combination of B7.1-Db and scFv-4-1BBL in a time-shift setting. HT1080-FAP cells were incubated with scDbFAPCD3 (33 pM) for 1 h, followed by washing and the addition of PBMC. After 3 days, PBMC were transferred to a well with freshly seeded HT1080-FAP and cocultured for another 7 days. For restimulation purpose, HT108-FAP had been previously incubated with the scDb (33 pM), followed by washing. Costimulation was applied by the addition of B7.1-Db (10 nM) and scFv-4-1BBL (10 nM) either simultaneously during initial stimulation or during restimulation with the scDb. Alternatively, B7.1-Db was applied together with initial scDb-mediated stimulation followed by scDb-mediated restimulation in presence of scFv-4-1BBL. T cell proliferation (a) and expression of granzyme B (b), PD-1 (c) and CTLA-4 (d) was measured after 7 days via flow cytometry. [n = 3. Mean ± SD, One-way ANOVA, Tukey post test, *p < 0.05, **p < 0.01, ***p < 0.0001]