CiTE-mediated T-cell activation depends on crosslink to target cells and is enhanced compared with BiTE-like molecule. (A) CiTE- and sctb-induced upregulation of CD69 and CD25 on HD T cells in comparison with controls as analyzed by flow cytometry in the absence or presence of MOLM-13:PD-L1 cells after 96 hours and E:T ratio of 2:1. Bar charts show mean values of n = 3 independent experiments at 5 nM concentration with SEM as error bars. (B) Fold change of IFN-γ release in the presence of MOLM-13:PD-L1 compared with MOLM-13 cells at 5 and 0.5 nM protein concentrations. (C) Granzyme B release in the presence of MOLM-13:PD-L1 cells at 0.5 nM protein concentration. (D) IFN-γ release in the presence of MOLM-13:PD-L1 cells upon addition of CiTE, sctb, or BiTE-like molecule at 5 nM protein concentration and combination of BiTE-like molecule with PD-1/PD-L1 blocking agents at equimolar concentration. Cytokine and Granzyme B release were measured after 72 hours at an E:T ratio of 2:1 using nonstimulated HD T cells. Bar charts represent mean values of (B,D) n = 4-5 or (C) n = 4-6 independent experiments with SEM as error bars. For statistical analysis, unpaired Student t test with Welch correction was applied. *P < .05, **P < .01, ***P < .001.